University of California, San Diego

UNIVERSITY OF CALIFORNIA, SAN DIEGO

The Mechanism of TAT-Mediated Cellular Transduction:

Role of Glycans and Rab GTPases

A dissertation in partial satisfaction of the requirements for the degree

Doctor of Philosophy

Biomedical Sciences

Jacob Morris Gump

Committee in charge:

Professor Steven F. Dowdy, Chair Professor Jeffrey Esko Professor James Feramisco Professor Yitzhak Tor Professor Benjamin Yu

2009

Jacob Morris Gump, 2009

The Dissertation of Jacob Morris Gump is approved, and is acceptable in quality and form for publication on microfilm and electronically:

______

______Chair

UNIVERSITY OF CALIFORNIA, SAN DIEGO

2009

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DEDICATION

I dedicate this dissertation to my wife Meagan and my son Eli. Without your constant love and support, this would not have been possible. I love you both; you are the greatest joys of my life.

EPIGRAPH

"Science is a wonderful thing if one does not have to earn one's living at it."

— Albert Einstein

TABLE OF CONTENTS

SIGNATURE PAGE ………..……………………………………………………………….. III

DEDICATION ………………..……………………………………………………………… IV

EPIGRAPH ………………….……………………………………………………………… V

TABLE OF CONTENTS …………………………………………………………………….. VI

LIST OF ABBREVIATIONS ………………………………………………………………….. VIII

LIST OF FIGURES …………………………………………………………………………. XI

ACKNOWLEDGEMENTS …………………………………………………………………... XIII

VITA ………………………………………………………………………………………. XV

ABSTRACT OF THE DISSERTATION ….………………………………………….... ………XVIII

CHAPTER 1: INTRODUCTION ……………………………………………………………… 1

ABSTRACT …..…………………………………………………………………… 1 HISTORY …..…………………………………………………….……………… 2 GLYCANS …………………….………………………………..……..……..…… 5 MACROPINOCYTOSIS ...…….…………………………………..……..………… 7 UPTAKE AND TRAFFICKING BY RAB GTPASES …….….……….……………… 11 BIOPHYSICS OF MEMBRANE TRANSLOCATION …..…….……….……………… 13 REFERENCES ..………………………………………………………………….. 16

CHAPTER 2: TAT TRANSDUCTION: THE MOLECULAR MECHANISM AND THERAPEUTIC PROSPECTS……………………………....……………….…………………..… 29

ABSTRACT …..…………………………………………………………………… 29 GENERAL INTRODUCTION TO MACROMOLECULAR THERAPEUTICS …………… 30 MECHANISM OF TAT-MEDIATED TRANSDUCTION INTO CELLS ………………… 32 THERAPEUTIC PROSPECTS …………………………………………..………… 41 CONCLUDING REMARKS ………………………………………………….…….. 45 GLOSSARY ………………………………………………………………………. 47 REFERENCES ……………………………………………………………………. 48

CHAPTER 3: REVISED ROLE FOR GLYCANS IN TAT-MEDIATED TRANSDUCTION…..…. 58

ABSTRACT…..………………………………………………………………….. 58 INTRODUCTION …………………………………………………………………. 59 EXPERIMENTAL PROCEDURES …………………………………………………. 62 RESULTS ………………………………………………………………………. 67 DISCUSSION …………………………………………………………………... 71 FIGURES ……………………………………………………………………….. 75 REFERENCES ………………………………………………………………….. 81

CHAPTER 4: TAT PROTEIN AND PEPTIDE TRANSDUCTION OCCURS BY ACTIVATING RAC1-DEPENDENT MEMBRANE RUFFLING AND RAB5 & RAB34-MEDIATED MACROPINOCYTOSIS .……………………………….…………………………. 86

ABSTRACT…..………………………………………………………………….. 86 INTRODUCTION ………………………………………………..……………….. 87 EXPERIMENTAL PROCEDURES ………………………………………………… 90 RESULTS ……………………………………………………………………… 96 DISCUSSION …………………………………………………………………….. 99 FIGURES ……………………………………………………………………….. 103 REFERENCES …………………………………………………………………. 108

CHAPTER 5: DISCUSSION ……………………………………………………………….. 114

REFERENCES …………………………………………………………………. 121

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LIST OF ABBREVIATIONS

Arf6 - Actin Remodeling Small GTPase

ATP - Adenosine Triphosphate

Bcl-XL - B-cell Lymphoma Extra-Large Protein

CCD - Charge-Coupled Device

CDC42 - Member of the Rho Family of Small GTPases

CHO - Chinese Hamster Ovary Derived Cell Line

CMV - Cytomegalovirus Promoter

CPP - Cell Penetrating Peptide

Cre - Cyclization Recombination Protein

DNA - Deoxyribonucleic Acid

EDTA - Ethylenediaminetetraacetic Acid

EGFP - Enhanced Green Fluorescent Protein

FACS - Fluorescent-Activated Cell Sorting

FITC - Fluorescein Isothiocyanate

FGF2 - Fibroblast Growth Factor 2 (Basic FGF)

FSC - Forward Scatter

GAG - Glycosaminoglycan

GDNF - Glial Cell Line-Derived Neurotrophic Factor

GTP - Guanosine Triphosphate

GTPase - GTP Hydrolysing Enzyme

HIV - Human Immunodeficiency Virus

HPLC - High Performance Liquid Chromatography

HS - Heparan Sulfate

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Hsp90 - Heat Shock Protein 90 kDa

HSPG - Heparan Sulfate Proteoglycan kDa - Kilodalton loxP - Locus of X-Over P1 Recombination Site

LTR - Long Terminal Repeat

MPG - PTD from N-methylpurine DNA glycosylase Protein p53 - Tumor Protein 53 kDa Transcription Factor

PAK1 - p21-Activated Kinase

PBS - Phosphate Buffered Saline

PDGF - Platelet-Derived Growth Factor

PG - Proteoglycan

PI3K - Phosphatidylinositide-3 Kinase

PKC - Protein Kinase C

PLC-γ - γ Phospholipase C Enzyme

PMA - Phorbol 12-myristate 13-acetate

PTD - Protein Transduction Domain

R8 - Octaarginine Peptide (RRRRRRRR)

Rab - Small GTPases with Roles in Endo & Exocytosis

Rac - Subfamily of Rho GTPases

Ras - Canonical Small GTPase

RNA - Ribonucleic Acid

RNAi - RNA Interference

SA - Sialic Acid siRNA - Small Interfering RNA

SOD - Superoxide Dismutase

SPPS - Solid Phase Peptide Synthesis

Src - Tyrosine Kinase (from Sarcoma)

SSC - Side Scatter

Tat - HIV Transactivator of Transcription Protein

TAT - Transduction Domain from HIV Tat Protein

TMR - Tetramethylrhodamine

WGA - Wheat Germ Agglutinin

LIST OF FIGURES

Chapter 3

Figure 3.1 TAT peptide cell association and cell surface binding in glycan-

deficient cells ………………. ………………………………………….. 75

Figure 3.2 TAT fusion protein cell association and cell surface binding in glycan-

deficient cells ………………. ………………………………………….. 76

Figure 3.3 TAT-Cre transduction occurs in the absence of glycosaminoglycans

and sialic acids in CHO parental and glycan-deficient cell lines.…. 77

Figure 3.4 Macropinocytotic inhibitors and TAT-Cre transduction in glycan-

deficient cells .………………………………………………………….. 78

Figure 3.5 TAT-induced macropinocytotic fluid-phase uptake is intact in glycan-

deficient cells .…………………………………………………………... 79

Figure 3.6 Enzymatic depletion of heparan sulfate and sialic acids does not impair

TAT transduction in parental or glycan-deficient cells despite efficient

removal of glycans.…………………………………………………….. 80

Chapter 4

Figure 4.1 TAT PTD activates Rac1 and TAT transduction is Rac1-dependent

……………..…………………..………………………..……………….. 103

Figure 4.2 TAT PTD induces Rac1-dependent membrane ruffling and

macropinocytosis …………………………………………………….. 104

Figure 4.3 GFP-Rab34 localizes to TAT-induced membrane ruffles and Rab34

Q111L expression alters TAT transduction and macropinocytosis.. 105

Figure 4.4 TAT PTD traffics through Rab5 endosomes and TAT transduction is

altered in cells expressing Rab5 Q79L …...... ……….. 106

Figure 4.5 Rab5 Q79L expression impairs TAT induction of macropinocytosis with

no effect on transferrin uptake …………………………….………….. 107

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ACKNOWLEDGEMENTS

First, I would like to thank my thesis advisor, Dr. Steven F. Dowdy for the opportunity to join his dynamic and consistently interesting lab. I thank Steve for the many scientific and life lessons learned in his lab. Among these are the value of positive thinking especially with respect to your project and the value of “just doing” an experiment you are positive won’t work. I also thank Steve for his generous support, especially in hard times, I am greatly indebted.

I would also like to thank the current and former members of the Dowdy lab.

Especially all the β side members who have helped, supported, pretended to listen and drank beer with me when appropriate. In particular, I thank Bryan Meade and

Gary Shapiro for thoughtful arguments and discussions on music, the existence of free will, Bush, lab gossip and occasionally our work. Their scientific and personal insights will remain with me forever. Other β’s of note who helped guide my scientific maturation are Aki, Hiro (not tight pants), Scott, Donger and Britta. Thanks to all.

Thanks to Gina and Leanne in the BMS office who help take the BS out of

BMS. I have enjoyed the fun times we have had together (especially at the retreat!).

Their help from beginning to end has been invaluable.

Thanks to Baja, our weekend sojourns to her beautiful and rugged coast brought fiery adventure and renewed vigor.

I thank Meagan, my wife and the love of my life, for the sacrifices she made to support my graduate career. I know it was not easy for her to postpone her dreams so that I could be miserable for 6 years. In return, I have given her my first born son.

A special thanks to my parents and my sister for their love and support, they made me who I am.

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The text of Chapter 2 is a reprint of the material as it appears in Trends in

Molecular Medicine, 2007, Vol 13, No 10, 443-448. Jacob M. Gump and Steven F.

Dowdy. The dissertation author was the primary author of this article.

Chapter 3, in part has been submitted for publication of the material. Jacob

M. Gump and Steven F. Dowdy. The dissertation author was the primary investigator and author of this material.

Chapter 4, in part is currently being prepared for submission for publication of the material. Jacob M. Gump and Steven F. Dowdy. The dissertation author was the primary investigator and author of this material.

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VITA EDUCATION:

2002-2009 Ph.D., Biomedical Sciences Program Department of Cellular and Molecular Medicine, University of California San Diego, School of Medicine, La Jolla, CA. Graduate Advisor: Professor Steven F. Dowdy, Ph.D.

1992-1997 B.A., Department of EPO Biology, University of Colorado, Boulder, CO.

1996-1997 Academic Exchange Program, Lancaster Univ., Lancaster, England.

PROFESSIONAL EXPERIENCE:

2003-2009 Graduate Research, Laboratory of Steven F. Dowdy, Howard Hughes Medical Institute, Department of Cellular and Molecular Medicine, University of California San Diego, School of Medicine, La Jolla, CA. Research Project: Mechanism of TAT-Mediated Transduction into Cells.

1998-2001 Research Technician, Laboratory of Stephen P. Hunger, Department of Pediatrics, Hematology/Oncology Laboratories, University of Colorado Health Sciences Center, Denver, CO. Research Project: p53 Mutation and p16 Inactivation in Acute Lymphoblastic Leukemia.

1997 Undergraduate Research, Laboratory of Rob Roy Ramey, Department of EPO Biology, University of Colorado, Boulder, CO. Research Project: Molecular Phylogenetic Analysis of Mammalian PrP Proteins.

1996 Undergraduate Research, Laboratory of Richard Olmstead, Department of Botany, University of Washington, Seattle, WA. Research Project: Phylogenetic Analysis of Boraginaceae Based on Chloroplast Gene Sequences.

HONORS:

1997 Howard Hughes Medical Institute Undergraduate Research Fellowship, Department of EPO Biology, University of Colorado, Boulder, CO. 1996-1997 Study Abroad Scholarship, University of Colorado at Boulder & Lancaster University, Lancaster, England, U.K. 1996 National Science Foundation Undergraduate Research Fellowship, Department of Botany, University of Washington, Seattle, WA.

TEACHING:

2007 Guest Lecturer, Graduate Seminar in Biomedical Research, Biomedical Sciences Program, University of California, San Diego, La Jolla, CA. 2007 Course Coordinator, Graduate Seminars in Cancer Biology, Department of Cellular and Molecular Medicine, UC San Diego, La Jolla, CA. 2005 Teaching Assistant, Undergraduate Pharmacology, Department of Biology, University of California, San Diego, La Jolla, CA.

COMMITTEES:

2006 Biomedical Sciences Retreat Committee, UC San Diego. 2004-2005 Biomedical Sciences Program Admissions Committee, UC San Diego. 2003-2004 Student-Faculty Liaison, Biomedical Sciences Program, UC San Diego.

RESEARCH PRESENTATIONS:

Gump J.M. & Dowdy S.F., 2007. Role of Glycosaminoglycans and Sialic Acids in TAT-Mediated Transduction. Biomedical Sciences Program Retreat, University of California San Diego.

Gump J.M. & Dowdy S.F., 2006. Mechanism of TAT-Mediated Transduction. Department of Cellular and Molecular Medicine Seminar, University of California San Diego.

Gump J.M. & Dowdy S.F., 2004. Purification, RNA Binding and Transduction of TAT- Argonaute2 Fusion Protein. Biomedical Sciences Program Graduate Seminar, University of California San Diego.

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PUBLICATIONS:

Gump, J.M. & Dowdy, S.F. Rab GTPases mediate macropinocytosis and cellular transduction of TAT peptides and fusion proteins. In preparation.

Gump, J.M. & Dowdy, S.F. TAT-mediated transduction of proteins and peptides occurs in the absence of extracellular glycosaminoglycans and sialic acids. Submitted.

Zhong, C.H., Prima, V., Liang, X., Frye, C., McGavran, L., Meltesen, L., Wei, Q., Boomer, T., Varella-Garcia, M., Gump, J.M., Hunger, S.P. E2A-ZNF384 and NOL1-E2A fusion created by a cryptic t(12;19)(p13.3; p13.3) in acute leukemia. Leukemia. 2008 Apr;22(4):723-9.

Gump, J.M. & Dowdy, S.F. TAT transduction: the molecular mechanism and therapeutic prospects. Trends Mol Med. 2007 Oct;13(10):443-8. Review.

Gump, J.M., McGavran, L., Wei, Q., Hunger, S.P. Analysis of TP53 mutations in relapsed childhood acute lymphoblastic leukemia. Journal of Pediatric Hematology/Oncology. 2001 Oct;23(7):416-9.

Meech, S.J., McGavran, L., Odom, L.F., Liang, X., Meltesen, L., Gump, J.M., Wei, Q., Carlsen, S., Hunger, S.P. Tropomyosin 4 (TPM4) is fused to ALK by a t(2;19)(p23;p13) in an unusual ALCL with the immunophenotype and functional properties of a natural killer cell malignancy. Blood. 2001 Aug 15;98(4):1209-16.

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ABSTRACT OF THE DISSERTATION

The Mechanism of TAT-Mediated Cellular Transduction: Role of Glycans and Rab GTPases

Jacob Morris Gump

Doctor of Philosophy in Biomedical Sciences

University of California, San Diego, 2009

Professor Steven F. Dowdy, Chair

TAT peptide transduction domain (PTD)-mediated cellular uptake permits the delivery of therapeutic macromolecules into cells in vitro and in vivo. Further understanding of the molecular players responsible for transduction will allow for enhanced efficiency and specificity of this tool, in order to take advantage of the enormous potential it holds. To this end, I have sought to elucidate the roles of glycans expressed on the cell surface and small GTPases in the process of macropinocytotic uptake of transducible peptides and proteins. Glycans, heparan sulfate proteoglycans in particular, have been postulated to mediate transduction by a variety of potential mechanisms. I have found, by multiple measures, that transduction occurs efficiently in the absence of glycosaminoglycans and sialic acids.

My data preclude the conclusion that TAT-mediated transduction is dependent on

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heparan sulfate. Instead they support a model where glycans (through charge based interactions) increase the amount of TAT that binds to the cell surface and is therefore transduced, but are mechanistically dispensible for PTD-mediated uptake.

Uptake of TAT PTD is known to occur via induction of macropinocytotic uptake, followed by escape from macropinocytic vesicles into the cytoplasm. However, the molecular basis for this process remains inadequately defined. I have found that TAT induces Rac1-dependent membrane ruffling and macropinocytosis. In addition, actin within TAT-induced membrane ruffles colocalized with Rab34, a small GTPase previously implicated in macropinocytosis. Furthermore, overexpression of Rab34 and Rab5 mutants altered the efficiency of transduction and macropinocytosis of

TAT. Together, my data support a model whereby TAT induces membrane ruffling and macropinocytosis via Rac1 and Rab34, independent of its binding to glycans.

These macropinosomes are then trafficked through Rab34 and Rab5-positive endosomal compartments. This knowledge further strengthens the role of macropinocytosis in TAT transduction and gives some of the first indications about trafficking and endocytic markers associated with TAT macropinosomes. Further study of the induction and trafficking of TAT macropinosomes may allow us to increase transduction efficiency by either enhancing the induction of macropinocytotic uptake or altering trafficking of macropinosomes to enhance endosomal escape.

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CHAPTER 1:

INTRODUCTION

Protein transduction refers to the ability of certain, primarily arginine-rich or amphipathic, peptides to gain entry to cells from the surrounding extracellular milieu.

These peptides are referred to interchangeably as protein (or peptide) transduction domains (PTD) or cell penetrating peptides (CPP). TAT and other PTDs have the potential to drastically expand the possible molecules available as therapeutics through their ability to transduce large macromolecules into cells. This ability to circumvent Lipinski’s rule of 6 has allowed researchers to deliver macromolecules including peptides, DNA, full-length proteins and siRNAs into cells in-vitro and in preclinical in-vivo disease models (Lipinski, Lombardo et al. 2001). PTD-mediated delivery has been used to successfully treat a number of diseases in mice, to deliver siRNAs and full-length proteins into stem cells and clinical trials are underway using

PTDs as drug delivery vehicles. The ability to use macromolecules therapeutically is a true paradigm shift; it will allow us to take advantage of genomic and structural data to cure diseases with specificity and genetic adaptability impossible using small molecule therapeutics. In addition, the ability to deliver siRNAs and proteins may allow us to therapeutically reprogram stem cells, or even a patient’s cells, in a non- genetic manner that holds as much promise as gene therapy once did, without the tragic shortcomings of transferring genetic material.

1 2

HISTORY

The HIV TAT protein transduction domain is one of several peptides with the unusual and useful ability to transport itself, and cargo linked to it, into cells. The ability of the full length HIV Tat protein (I will use upper case TAT to refer to the transduction domain and Tat to refer to the HIV protein throughout this document to avoid confusion) to transduce cells was first recognized by Frankel & Pabo in the late

1980s (Frankel and Pabo 1988). They showed that when Tat protein was applied to cells exogenously, it entered cells and was able to transactivate the viral LTR. Further characterization of this phenomenon allowed researchers to map this capability to a short basic domain of the protein, amino acids 38-58 (Mann and Frankel 1991). This has been pared down to an even smaller peptide, amino acids 49-57, that is sufficient for optimal delivery and this is what we now refer to as the TAT PTD (Vives, Brodin et al. 1997). Some researchers have indicated that full length Tat protein is secreted by

HIV infected cells and that the ability of full length HIV Tat to transduce cells and activate viral transcription is physiologically important in HIV infection, though this remains controversial. However, the importance of TAT lies not only in its normal physiologic function, but in its ability to ferry other cargo into cells in vitro and in vivo.

My work has focused on the ability of TAT PTD peptide to transduce large cargoes into cells and the mechanism used to accomplish this feat.

Early reports on the mechanism of full-length TAT protein transduction indicated that the route of delivery was endocytotic, based on visualization of cells, and the observations that entry was inhibited by incubation at 4 °C and enhanced by treatment with chloroquine (Mann and Frankel 1991). However, this observation was essentially forgotten and later mechanistic work on TAT and other PTDs indicated

that transduction occurred in an energy-independent manner and that these peptides simply permeated the outer cell membrane and entered the cytoplasm (Derossi, Joliot et al. 1994; Derossi, Calvet et al. 1996; Vives, Brodin et al. 1997; Rothbard,

Garlington et al. 2000; Futaki, Suzuki et al. 2001; Suzuki, Futaki et al. 2002).

However, the experimental techniques leading to these conclusions are now known to be flawed (Richard, Melikov et al. 2003; Brooks, Lebleu et al. 2005). They were based primarily on the microscopic observation of fixed cells showing that PTD peptides were present inside cells even when the cells were treated with PTD at 4 °C.

This phenomenon is an artifact of cell fixation; PTDs do not enter cells simply by passively crossing the membrane. Fixing cells, especially with methanol or acetone, causes redistribution of TAT and other PTDs and can drastically alter the subcellular localization of these peptides and their cargoes. In addition, PTD molecules bound to the outside of cells redistribute to the inside of the cell during fixation. Flow cytometry data indicating direct membrane permeation is also suspect because even after extensive washing large amounts of peptide remain bound to the cell surface leading to a false positive signal (Richard, Melikov et al. 2003). Current protocols indicate both protease digestion and heparin washing to remove this “cell associated” peptide before FACS (Kaplan, Wadia et al. 2005). More recent data support the original theory postulated by Frankel and Pabo that TAT and other PTDs enter cells actively by endocytosis and that this uptake is stimulated by these peptides.

Despite these criticisms with regards to the technical aspects of measuring

PTD uptake, most researchers still rely on the visualization or FACS of labeled PTDs as their primary measure of efficiency and mechanistic action. We prefer to measure, in addition to cell association which is a good approximation for most purposes,

transduction using a phenotypic assay whereby the cargo being delivered is either an enzyme or enzyme substrate that must enter the cytoplasm or nucleus of the cell to give a measurable response. To this end, we have been researching the use of enzyme substrates or enzyme complementation peptides with only limited success.

Most readily assayable enzyme substrates are cell permeable on their own (i.e. luciferin) and the complementation assays we have attempted are not robust enough to produce reliable results. Our most successful endeavor in this arena is the use of

TAT PTD linked transducible Cre enzyme (Abremski and Hoess 1984; Lin, Jo et al.

2004; Wadia, Stan et al. 2004). Using TAT-Cre based assays in live cells, we are able to determine the actual efficiency of transduction in a system that drastically reduces the chance of false positive results and avoids most of the caveats of other measures of transduction. In order to get a positive result using TAT-Cre, the enzyme must bind to the cell, be taken up by endocytosis, escape the endosome (in an active state) and transit to the nucleus to recombine loxP sites. This is, by far, the most accurate measure of whether transduction has occurred in a cell that we have found to date.

However, the readout is binary – either the cell is green or not – therefore, this assay does not allow us to measure the amount of transduction that has occurred in individual cells.

GLYCANS

Early observations of full-length Tat protein showed that uptake could be inhibited by the presence of sulfated glycans in solution and that the protein bound avidly to heparin and heparan sulfate (HS) (Mann and Frankel 1991; Albini, Benelli et al. 1996; Rusnati, Coltrini et al. 1997). However, entry into cells was not affected by pre-treatment with neuraminidase or heparinase to remove extracellular sialic acid

(SA) or HS, indicating that at least these two major types of sulfated glycans were unnecessary for transduction (Mann and Frankel 1991). Skip forward ten years and

Tyagi et al find that HS is necessary for transduction of Tat protein by showing that cells genetically deficient for glycosaminoglycans (GAGs), including HS, show reduced (thought not abolished) uptake and transactivation (Tyagi, Rusnati et al.

2001). This apparent contradiction is perplexing and may be explained by the different cells and systems used to test these hypotheses. Further work by some of the same researchers indicated that, contrary to what was seen with Tat protein, the

TAT basic peptide entered cells in an energy-independent manner that did not depend on HS or endocytosis (Silhol, Tyagi et al. 2002). These findings further complicate the matter and are difficult to interpret. Several other publications have confirmed that transduction of full length Tat protein is dependent on HS proteoglycans (HSPGs) (Chang, Samaniego et al. 1997; Liu, Jones et al. 2000). In addition, several groups have indicated that multiple cationic PTDs, including TAT, depend on HSPGs for their uptake (Suzuki, Futaki et al. 2002; Console, Marty et al.

2003; Ziegler and Seelig 2004; Goncalves, Kitas et al. 2005). Glycosaminoglycan- deficient cells were also found to be impaired in their ability to activate actin remodeling in response to treatment with polyarginine PTD (Nakase, Tadokoro et al.

2007).

In light of the above results, we obtained HS deficient cell lines with the goal of using them as transduction null cells to dissect the molecular mechanism of transduction. In our hands, the cells had lower transduction efficiency, but, to our surprise, they were not refractory to transduction of labeled TAT peptides or TAT-Cre protein (Gump and Dowdy 2009). These results are elaborated extensively in Chapter

3 of this dissertation. Several other researchers have come to similar, conflicting conclusions, including the original descriptions of Tat transduction (Mann and Frankel

1991; Silhol, Tyagi et al. 2002; Violini, Sharma et al. 2002; Console, Marty et al.

2003). While many contradictions exist with regard to the role of HSPGs in PTD entry, they appear, at the minimum, to aid in transduction. Further analysis of transduction pathways and their absence or existence in glycan-deficient cells will allow us to more adequately define the role of glycans in transduction of TAT and other PTDs.

MACROPINOCYTOSIS

Macropinocytosis was originally observed by Lewis in 1931 as the formation of large vesicles from the base of membrane ruffles (Lewis 1931). Macropinocytosis is defined by both its morphological and mechanistic characteristics. Macropinosomes are large (0.2 – 5 µM) endosomes that form from actin protrusions (ruffles) on the cell surface which close and engulf extracellular fluid. This process is not guided directly by ligands but can be induced by extracellular growth factors or mitogens.

Macropinocytosis is known to be inhibited by amiloride and its analogs, which have no effect on other forms of endocytosis. Because of the inability to track and observe macropinosomes via receptor-ligand interactions, macropinocytosis remains loosely defined and one of the most poorly understood forms of endocytosis (Kerr and

Teasdale 2009).

The fate of macropinocytotic vesicles is a subject of some controversy and their trafficking may be cell type specific. Some researchers have reported that macropinosomes traffic via the endo-lysosomal pathway whereas others have described their contents being recycled back to the cell surface (Swanson 1989;

Racoosin and Swanson 1992; Racoosin and Swanson 1993; Hewlett, Prescott et al.

1994; Swanson and Watts 1995; Schnatwinkel, Christoforidis et al. 2004). However, macropinosomes use many of the same endocytic components as more typical ligand-receptor-mediated endocytosis, it is therefore likely that their components are sorted, recycled and degraded in a similar manner (Lanzetti, Palamidessi et al. 2004;

Swanson 2008; Kerr and Teasdale 2009). The size of macropinosomes also makes them particularly susceptible to usurpation by microorganisms; the macropinosome is

therefore not likely to be more hospitable than smaller clathrin or caveolin endosomes which are physically incapable of endocytosing all but small particles.

Membrane ruffling leading to macropinocytosis is mediated by actin polymerization following induction by growth factors or other stimulation. This process is activated by the Rho GTPase Rac that activates and coordinates the formation of planar sheet-like protrusions of the cell membrane known as membrane ruffles

(Nobes and Marsh 2000; Schafer, D'Souza-Schorey et al. 2000; West, Prescott et al.

2000). Ruffling activity is either constitutive as seen in many transformed cells or can be induced by growth factors or mitogens (Bar-Sagi and Feramisco 1986; Racoosin and Swanson 1989; Sallusto, Cella et al. 1995). Membrane ruffles precede and are necessary for the formation of macropinosomes, but the two processes are regulated independently (Li, D'Souza-Schorey et al. 1997). Most membrane ruffles form and recede into the cytoplasm based on the variable and reversible activities of GTPases and inositide kinases and phosphatases. These ruffles sometimes form circular cup- like extravasations; the rim of the cup then contracts and fuses in the third dimension to form a macropinosome (Swanson 1989; Swanson and Watts 1995; Araki, Johnson et al. 1996; Ellerbroek, Wennerberg et al. 2004; Swanson 2008). This process is very similar to phagocytosis but is self-guided rather than being guided by the particle it is ingesting (Swanson 2008).

Formation of macropinosomes requires concerted activity of many intracellular signaling networks to coordinate such an exquisitely complicated process, that occurs, unlike most endocytic processes, in the absence of any template or scaffold. The small GTPases Rac1, cdc42, RhoG and Arf6 are involved in

macropinosome formation where they coordinate actin-dependent ruffling and cup formation, in addition to coordinating myosin contractile activity necessary for inward movement of the cup before closure (Garrett, Chen et al. 2000; Nobes and Marsh

2000; Schafer, D'Souza-Schorey et al. 2000; West, Prescott et al. 2000; Ridley 2001;

Anton, Saville et al. 2003). PI3K and Pak1 are also necessary for macropinocytosis and play roles in actin polymerization and in the final fissure of macropinosomes from the cell membrane, a process which, unlike other forms of endocytosis occurs independent of dynamin (Araki, Johnson et al. 1996; Cox, Tseng et al. 1999;

Edwards, Sanders et al. 1999; Ridley 2001; Yang, Vadlamudi et al. 2005; Mercer and

Helenius 2008). Several Rab GTPases appear to regulate and facilitate macropinosome formation and trafficking. These include Rab5, Rab7 and Rab34; these are discussed below and at length in chapter 4 of this dissertation.

The classic definition of macropinocytosis as a mechanism for non-selective uptake of extracellular fluid is undoubtedly insufficient. Macropinocytosis has a crucial role in antigen uptake and presentation, and is known to mediate the uptake of several endogenous ligands (Sallusto, Cella et al. 1995; Nobes and Marsh 2000;

Tkachenko, Lutgens et al. 2004). Macropinocytosis is also responsible for the uptake of several intracellular pathogens; this realization has led to a recent burst in research and has helped greatly to elucidate the mechanism of macropinocytosis. The mechanisms used by pathogens to expropriate actin rearrangement and macropinocytosis have been reviewed recently (Rottner, Stradal et al. 2005; Mercer and Helenius 2009). The uptake and activity of several viruses, including adenovirus, coxsackievirus and Kaposi’s sarcoma-associated herpesvirus, depend on macropinocytosis (Meier, Boucke et al. 2002; Coyne, Shen et al. 2007; Amstutz,

Gastaldelli et al. 2008; Mercer and Helenius 2008; Raghu, Sharma-Walia et al. 2009).

Likewise, the entry of Salmonella, Legionella and E. coli appear to occur via macropinocytosis (Alpuche-Aranda, Racoosin et al. 1994; Watarai, Derre et al. 2001;

Laakkonen, Makela et al. 2009).

The crucial role of macropinocytosis in uptake of TAT and other PTDs was first reported by our lab and has subsequently been confirmed by others (Nakase,

Niwa et al. 2004; Wadia, Stan et al. 2004; Wang and El-Deiry 2004; Kaplan, Wadia et al. 2005; Melikov and Chernomordik 2005; Snyder, Saenz et al. 2005; Khalil, Kogure et al. 2006; Magzoub, Sandgren et al. 2006). TAT PTD has been shown to induce, via an unknown mechanism, Rac-dependent actin remodeling, membrane ruffling and macropinocytotic uptake (Nakase, Niwa et al. 2004; Gerbal-Chaloin, Gondeau et al.

2007). This is discussed in more depth in Chapter 4 of this dissertation. Alternately, several published reports indicate that other forms of endocytosis may mediate PTD uptake and transduction (Ferrari, Pellegrini et al. 2003; Richard, Melikov et al. 2005).

These entry pathways may not be mutually exclusive and it may be that PTD uptake occurs via several endocytic pathways simultaneously, or may depend on the cargo and cell type being studied.

UPTAKE AND TRAFFICKING BY RAB GTPASES

The activity of small GTPases is crucial for all forms of endocytosis, including

TAT PTD-induced macropinocytosis. TAT PTD is known to induce its own uptake by macropinocytosis but the trafficking and formation of these endosomes is poorly defined. Several small GTPases are postulated to control and mediate macropinocytosis and macropinosome trafficking. Rabs are involved in a multitude of steps in endo and exocytic pathways, including vesicle formation, motility, targeting, docking and fusion, in addition to remodeling of membranes (Albini, Benelli et al.

1996; Nielsen, Severin et al. 1999; Somsel Rodman and Wandinger-Ness 2000;

Zerial and McBride 2001). Conversion of endosomes from one Rab to another may also regulate their progression from one compartment to another (Rink, Ghigo et al.

2005). Among the most well studied and most important is Rab5, which is involved in all forms of endocytosis. Rab5 is associated with nascent endocytic vesicles and early endosomes and controls both ligand-dependent and independent endocytosis

(Bucci, Parton et al. 1992; Stenmark, Valencia et al. 1994; Bucci, Lutcke et al. 1995).

Rab5 plays an important role in endosome fusion and conversion to and from early endosomes (Gorvel, Chavrier et al. 1991; Stenmark, Parton et al. 1994; Roberts,

Barbieri et al. 1999). In addition to Rab5’s activities as a mediator of endosome movement and fusion, Rab5 is known to regulate the activity of Rac1 whose activity is required for ruffling and macropinocytosis (Palamidessi, Frittoli et al. 2008). Rab5 mutants inhibit Rac activation, membrane ruffling and fluid phase uptake. Other Rabs that have been implicated in macropinocytosis are Rab7 and Rab34. Rab7 is a late endosomal marker that associates with macropinosomes (Racoosin and Swanson

1993; Kerr, Lindsay et al. 2006; Lim, Wang et al. 2008). Rab34 has been shown to colocalize to membrane ruffles and nascent macropinosomes, and expression of a

dominant negative mutant inhibits macropinocytosis (Chen, Han et al. 2003; Sun,

Yamamoto et al. 2003; Coyne, Shen et al. 2007). I have explored the function of

Rabs in macropinocytosis of TAT PTD; more information can be found in Chapter 5 of this document.

BIOPHYSICS OF MEMBRANE TRANSLOCATION

The finding that TAT PTD transduction occurs via endocytosis does not negate the ability or necessity of PTDs to translocate through biological membranes, it simply moves the location of membrane translocation from the outside of the cell to the inside of an endocytic vesicle (Fischer, Fotin-Mleczek et al. 2005; Melikov and

Chernomordik 2005). The contents of endocytic vesicles are not merely dumped into the cytoplasm of the cell, myriad mechanisms exist to direct and actively uptake contents that are desired, while degrading those that are potentially harmful to cells.

The many pathogens which enter cells via macropinocytosis use methods to subvert normal degradative pathways. PTD uptake must also avoid endo-lysosomal destruction, but the mechanisms used to avoid this fate are unknown. Several studies have indicated that PTDs enter cells via retrograde transport into the golgi while others support the idea that PTD-containing vesicles are transported directly to the nucleus; there is little evidence to support either of these hypotheses (Fischer, Kohler et al. 2004; Noguchi and Matsumoto 2006). The generally held view in the field today is that TAT and most other PTDs are released from endocytic vesicles by disruption of normal membrane topology through biophysical perturbation.

Early research on the membrane permeability of TAT and other PTDs maintained that “cell permeation” occurred at the outer membrane of the cell and there is evidence to support this ability for certain, primarily toxic, peptides. While this is not likely the case with most PTDs, the changing environment within endocytic vesicles may promote membrane disruption and escape into the cytoplasm (Richard,

Melikov et al. 2003). There are several lines of evidence to support endocytic release as the mechanism of translocation into cells. Endosomal disruption is known to

increase delivery of PTDs, as does the addition of membrane disrupting compounds and the addition of membrane disrupting domains to PTDs (Caron, Quenneville et al.

2004; Wadia, Stan et al. 2004; Sakai, Takeuchi et al. 2005; Takeuchi, Kosuge et al.

2006; El-Sayed, Futaki et al. 2009). In addition, pH changes that accompany endosome trafficking are known to facilitate interaction of TAT and other PTDs with model membranes (Koch, Reynolds et al. 2003; Potocky, Menon et al. 2003; Fischer,

Kohler et al. 2004; Magzoub, Pramanik et al. 2005).

Decreasing pH is not the only endosomal change that increases the interaction of PTDs with membranes. The internal and membrane constituents of endocytic vesicles are constantly changing as the vesicles traffic and fuse with other compartments. Among the changes which are likely to be important for escape of

TAT from macropinosomes are the pH, peptide concentration, vesicle size, membrane lipid makeup, and membrane potential. High concentrations of PTD peptides are known to cause membrane disruption even at the cell surface if the concentrations used are high enough. Additionally, TAT and other PTDs are able to disrupt the membrane topology of several model membrane systems that mimic endosomal escape (Thoren, Persson et al. 2004; Curnow, Mellor et al. 2005; Thoren,

Persson et al. 2005; Afonin, Frey et al. 2006). Membrane potential may also play a role in the ability of cationic PTDs to transduce cell membranes (Terrone, Sang et al.

2003; Rothbard, Jessop et al. 2004).

The role of charge in membrane transduction is downplayed by the fact that replacement of arginine by lysine severely reduces the efficiency of PTDs. However, this underscores the importance of arginine residues for transduction and

experiments using synthetic peptoids and dendrimers indicate that the guanidino moiety is crucial for transduction (Mitchell, Kim et al. 2000; Wender, Mitchell et al.

2000; Futaki, Suzuki et al. 2001; Futaki 2002; Luedtke, Carmichael et al. 2003;

Chung, Harms et al. 2004). The mechanism responsible for arginine’s importance may be its ability to form resonant bidentate hydrogen bonds with sulfate and phosphate ions on cell surface glycans and phospholipids. This bidentate complexation of guanidinium with anions appears to increase the lipophilicity of arginine-rich peptides and may enable them to transiently enter and/or disrupt biological membranes (Rothbard, Jessop et al. 2004; Perret, Nishihara et al. 2005;

Sakai, Takeuchi et al. 2005). Further supporting a role for transient membrane entrance or disruption is the presence of a point of diminishing returns in polyarginine peptide length with the optimal range being ~7-15 residues. The idea being that beyond a certain number of arginine residues, the affinity for the membrane becomes too high and the interaction is no longer transient. Together, these data support a mechanism whereby changes in pH (and/or other alterations) within endocytic vesicles favor a more intimate interaction between TAT and the cell membrane, this transiently forms an inverted micelle which causes the vesicle to spill some of its contents (PTD and cargo) into the cytoplasm.

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CHAPTER 2:

TAT TRANSDUCTION: THE MOLECULAR MECHANISM

AND THERAPEUTIC PROSPECTS

ABSTRACT

Research into the mechanism of protein transduction has undergone a renaissance in the last five years as many groups have sought to understand the behavior of these peptides in order to harness their enormous therapeutic and diagnostic potential. The field has benefited greatly from rigorous cell biological and biophysical study of the mechanism used by cell penetrating peptides to enter cells and deliver their cargo. The recent identification of fluid phase endocytosis as the mode of cellular entry for TAT and other protein transduction domains has enhanced our understanding of how transduction facilitates intracellular delivery. Many outstanding questions and contradictions still remain to be resolved in the field.

Nevertheless, the current body of work regarding the mechanism of uptake gives a much clearer picture of how these macromolecules enter cells and how we might enhance the bioavailability to take advantage them clinically.

GENERAL INTRODUCTION TO MACROMOLECULAR THERAPEUTICS

The plasma membrane presents a formidable barrier to the introduction of macromolecules into cells. For nearly all therapeutics to exert their effects, at least one cellular membrane must be traversed. Traditional small molecule pharmaceutical development relies on the chance discovery of membrane permeable molecules with the ability to modulate protein function. While small molecules remain the dominant therapeutic paradigm, many of these information-poor molecules suffer from lack of specificity, side effects, and toxicity. Through rational design based on molecular, cellular and structural data we can create information-rich macromolecules with protein-modulatory functions far superior to those of small molecules. However, the plasma membrane is impermeable to most molecules greater than 500 Daltons. The ability of cell penetrating peptides like TAT to cross the cell membrane and deliver macromolecular cargo in vivo will greatly facilitate the rational design of future therapeutic proteins, peptides and nucleic acids. Understanding the mechanism used by protein transduction domains to enter cells has already enhanced the efficiency of transduction and will facilitate further improvements in both delivery and therapeutic design.

The TAT cell penetrating peptide (CPP) or protein transduction domain

(PTD) was derived from amino acids 47-57 of the HIV Tat protein after it was shown that full length Tat protein could be taken up by cells and activate transcription of the viral genome (Mann and Frankel 1991; Fawell, Seery et al. 1994; Vives, Brodin et al.

1997). Since then, the TAT PTD and other CPPs have been shown to deliver cargoes as large as iron nanobeads and fluorescent quantum dots into cells in culture

(Josephson, Tung et al. 1999; Pittet, Swirski et al. 2006). TAT has also been used to

deliver large, active proteins into the cells of live mice and TAT fusion proteins and peptides have been used to treat mouse models of cancer, inflammation and other diseases (Snyder and Dowdy 2005; Wadia and Dowdy 2005). TAT has also been used to deliver phage encapsulated DNA to cells and liposome encapsulated DNA for gene expression in mice (Eguchi, Akuta et al. 2001; Glover, Lipps et al. 2005). It should also be noted that efficiency of transduction of TAT and other CPPs depends greatly on the cargo being transduced; this principle is most clearly demonstrated by difficulties in transducing large, anionic cargo like nucleic acids (Fischer, Fotin-

Mleczek et al. 2005; Tunnemann, Martin et al. 2006; Meade and Dowdy 2007). The above examples demonstrate the breadth of strategies where TAT has been used successfully for delivery in cell culture and in vivo. In this review we shall discuss recent findings concerning the mechanism of transduction and also review current and future applications of TAT and other CPPs in the delivery of macromolecular therapeutic agents.

MECHANISM OF TAT-MEDIATED TRANSDUCTION INTO CELLS

The mechanism used by these peptides to permeate cell membranes has been the subject of considerable study in recent years, as researchers have sought to understand the biology behind transduction. Early reports that TAT transduction occurred by a non-endocytic mechanism have largely been dismissed as artifactual although other CPPs may be taken up via direct membrane disruption (El-Andaloussi,

Holm et al. 2005; Fischer, Fotin-Mleczek et al. 2005; Fotin-Mleczek, Fischer et al.

2005; Gupta, Levchenko et al. 2005). The recent findings by our lab and others that transduction of TAT and other PTDs occurs via macropinocytosis has created a new paradigm in the study of these peptides. Enhanced knowledge of the mechanism of transduction has also enabled our group and others to improve transduction efficiency with the ultimate goal of clinical success.

The current model for TAT-mediated protein transduction is a multi-step process that involves binding of TAT to the cell surface, stimulation of macropinocytotic uptake of TAT and cargo into macropinosomes and endosomal escape into the cytoplasm. The first step, binding to the cell surface, is thought to be through ubiquitous glycan chains on the cell surface. Stimulation of macropinocytosis by TAT occurs by an unknown mechanism that may include binding to a cell surface protein or may also occur via proteoglycans or glycolipids. Uptake via macropinocytosis, a form of fluid phase endocytosis used by all cell types, is required for TAT and polyarginine transduction (Nakase, Niwa et al. 2004; Wadia, Stan et al.

2004). The final step in TAT transduction is escape from macropinosomes into the cytoplasm; this process is likely to be dependent on the pH drop in endosomes that,

along with other factors, facilitates a perturbation of the membrane by TAT and release of TAT and cargo to the cytoplasm.

Cell Surface Binding

The molecules that TAT binds on the cell surface to facilitate its uptake are not known. The highly charged arginine residues present within TAT are necessary for transduction and the substitution of any of these residues dramatically reduces the efficiency of transduction (Wender, Mitchell et al. 2000). Arginine differs from other cationic amino acids not only in its very high pI through resonance stabilization of the guanidino group, but also in its ability to form bidentate hydrogen bonds with sulfate, phosphate or carboxylate anions (Rothbard, Jessop et al. 2004). These interactions are likely to be important not only for cell surface interaction but also for membrane translocation. The ability of the arginine guanidino groups to interact strongly with these anions leaves open many possibilities for cell surface binding partners.

Interactions with acid regions of proteins, sulfated glycans, membrane phospholipid head groups or a combination of these may be responsible for macropinocytosis and membrane transduction of TAT.

TAT and other polycationic transduction domains are known to bind strongly to sulfated glycans like heparan sulfate and their presence in solution can compete

TAT from the cell surface and inhibit transduction (Rusnati, Coltrini et al. 1997;

Hakansson, Jacobs et al. 2001). Indeed, a role for cell-surface polyanionic glycan sugar chains in transduction of TAT is generally assumed. A fairly large body of evidence exists to support the role of glycans in transduction, however, a few papers have been published that dispute these conclusions (Silhol, Tyagi et al. 2002; Violini,

Sharma et al. 2002). In addition, recent data from our lab indicate that TAT-mediated transduction occurs in the absence of heparan sulfate, chondroitin sulfate and sialic acids (Gump & Dowdy, in prep).

Heparan and chondroitin sulfate glycans and sialic acids form a forest of negative charge that binds the highly basic TAT on the cell surface. These glycans are thought to both serve as a binding pool for TAT but also to facilitate its interaction with the membrane. Beyond the data to support the involvement of heparan sulfate in transduction, the theory that transduction occurs via heparan sulfate proteoglycans is favored for several reasons: first, a canonical ligand-receptor interaction between

TAT and its binding partner(s) is not likely as D amino acids and scrambled sequences seem to perform as well as wild-type (Derossi, Calvet et al. 1996; Wender,

Mitchell et al. 2000). Second, transduction appears to be independent of cell type and while different cells display different glycans, all cells are coated with these polysaccharides. Additionally, hydrogen bonding between TAT arginine guanidino groups with sulfate has been shown to increase the hydrophobicity of the peptide that could facilitate its interaction with the membrane (Rothbard, Jessop et al. 2004).

Multiple groups have concluded that heparan sulfate is required for transduction based on multiple readouts including fluorescently labeled peptides and proteins. These data show that in cells lacking or deficient in these glycans, transduction is reduced (Tyagi, Rusnati et al. 2001; Console, Marty et al. 2003).

However, the extent of this reduction is variable and depends on the assay; in many cases the amount of transduction seen is still well above background (Nakase,

Tadokoro et al. 2007). In order to conclude that heparan sulfate is required for

transduction it is necessary to use more robust, sensitive and quantitative assays to determine the extent of the decrease in transduction seen in glycan deficient cell lines and whether this decrease is truly glycan dependent.

The role of heparan sulfate and other proteoglycans in TAT macropinocytosis has not been fully explored. It is possible that glycans serve not only to bind TAT but also to stimulate its endocytosis. Several proteoglycans that have been shown to interact with TAT peptide and full length HIV TAT protein and these could serve as receptors for TAT and induce macropinocytosis (Argyris,

Kulkosky et al. 2004). In addition syndecan proteoglycans have been shown to be responsible for the uptake of FGF2 by macropinocytosis, indicating that proteoglycans could facilitate the uptake of TAT by macropinocytosis (Tkachenko,

Lutgens et al. 2004).

Role for Endocytotic Uptake in TAT Transduction

In one of the earliest studies, endocytosis was found to be responsible for uptake of HIV tat protein (Mann and Frankel 1991). However, subsequent studies on

TAT and other PTDs showed that uptake occurred at 4 °C in a manner independent of cellular metabolism. This led to the conclusion that these peptides and cargo were translocating directly through the plasma membrane. In contrast, recent data dispute these early mechanistic studies and indicate that transduction does not occur at 4 °C, depends on cellular ATP, cytoskeletal rearrangment and is an active rather than passive physical process (Richard, Melikov et al. 2003; Nakase, Niwa et al. 2004;

Fischer, Fotin-Mleczek et al. 2005; Fotin-Mleczek, Fischer et al. 2005; Kaplan, Wadia et al. 2005).

Macropinocytosis is a form of fluid phase endocytosis wherein actin protrusions (circular ruffles) fold in on the cell and uptake the surrounding medium.

Less is known about macropinocytosis relative to other well studied forms of endocytosis like the coated pit, clathrin and phagocytic pathways. Macropinocytosis is thought to occur in all cell types and because of the size of macropinosomes, large extracellular particles are taken up by the cells. This form of endocytosis can be inhibited by cytochalasinD, an actin polymerization inhibitor and is more specifically inhibited by amiloride and its analogs.

The evidence for macropinocytosis as the mechanism for TAT uptake is extensive. Data from our lab and others indicate, by a variety of measures, that TAT and other cationic PTDs promote their own uptake by macropinocytosis and that inhibitors of macropinocytosis abrogate transduction (Wadia, Stan et al. 2004;

Kaplan, Wadia et al. 2005). Importantly, TAT fusion proteins and peptides have been shown to induce the uptake of neutral dextran, a marker of fluid phase endocytosis

(Wadia, Stan et al. 2004; El-Andaloussi, Johansson et al. 2006). TAT stimulation of macropinocytosis is important because it indicates that these molecules are not just passively entering cells but are stimulating their own endocytic uptake. In the presence of inhibitors of macropinocytosis like cytochalasinD or amiloride, the transduction of TAT fusion proteins and TAT peptides is reduced to background levels (Wadia, Stan et al. 2004; Kaplan, Wadia et al. 2005). In addition, polyarginine transduction has been shown to be sensitive to amiloride and cytochalasinD further indication that macropinocytosis is responsible for the uptake of cationic PTDs

(Nakase, Niwa et al. 2004; Khalil, Kogure et al. 2006). The activity of the small

GTPase Rac is necessary for transduction, consistent with the role for Rac in the actin rearrangement necessary for macropinocytosis (Gerbal-Chaloin, Gondeau et al.

2007; Nakase, Tadokoro et al. 2007).

A role for clathrin or caveolae in endocytosis of CPPs has been demonstrated but its role in TAT uptake is unclear. Several groups have concluded that these pathways are at least partially responsible for the transduction of TAT and other PTDs (Fischer, Kohler et al. 2004; Richard, Melikov et al. 2005; Duchardt, Fotin-

Mleczek et al. 2007). Data from our lab indicate that TAT transduction occurs in cells lacking caveolae and that transduction is not inhibited by the presence of dominant negative Dynamin1, an inhibitor of clathrin-mediated uptake. While the particular type of endocytosis is a matter of some debate, it is generally agreed that endocytosis is a requisite step in transduction of TAT and many other PTDs (Richard, Melikov et al.

2003; Fischer, Kohler et al. 2004). A number of factors may contribute to the confusion and debate, among these are the particular PTD being studied, the cargo and the cell type (Richard, Melikov et al. 2005).

Endosomal Escape and Trafficking

The earliest and most basic mechanistic question about TAT protein transduction, and transducible peptides in general, has been how these peptides can cross the cell membrane. This is still the most important unsolved mystery in the field and while cellular and biophysical data abound, there is still no clear answer. Early mechanistic studies showing that membrane transduction was energy independent and could occur at 4 °C indicated a biophysical perturbation of the membrane was occurring and many studies ensued on the interaction of TAT with model membrane

systems (Magzoub, Kilk et al. 2001; Ziegler, Blatter et al. 2003; Goncalves, Kitas et al. 2006). Most researchers now concur that protein transduction occurs via endocytosis and in the absence of endocytosis, no uptake occurs (Console, Marty et al. 2003; Richard, Melikov et al. 2003; Fischer, Kohler et al. 2004; Nakase, Niwa et al.

2004; Wadia, Stan et al. 2004). However, this merely changes the site of membrane translocation from the cell surface to a vesicular compartment, because the inside of a vesicle is still essentially the outside of the cell. Endocytosis leaves the peptide

(and cargo) inside a vesicle that it must escape to enter the cytoplasm since most bioactive cargo must be in the nucleocytoplasmic space to exert an effect.

Escape of TAT from vesicles to the cytoplasm appears to be the rate limiting factor in the efficiency of transduction. The process depends on several factors that conform to what is known about the maturation of endocytic vesicles. First, the interaction of TAT with the membrane is enhanced by decrease in pH and, conversely, buffering the pH in endosomes inhibits transduction. Second, transduction may depend on changes in membrane potential that are known to accompany endocytosis. Lastly, changes in concentration of endosomal content as well as membrane lipid and protein components along the endosomal route may enhance transduction. These processes are thought to facilitate the interaction of

TAT with the membrane to disrupt it in such a way that TAT and attached cargo enter the cytoplasm.

Biophysical evidence has shed light on how TAT interacts with the membrane and how this interaction could facilitate endosomal escape. TAT has been shown to traverse the lipid bilayer of giant unilamellar vesicles but not large

unilamellar vesicles that are more rigid (Thoren, Persson et al. 2004). Additionally,

TAT and polyarginine PTDs can also promote the fusion of phospholipid vesicles in a manner that does not lead to leakage of the vesicle contents (Thoren, Persson et al.

2005). TAT has also been shown to induce a non-lamellar phase and rod-like inverted micelles in saturated phosphatidylcholine membranes (Afonin, Frey et al.

2006). These findings give important insight into the how TAT might escape from endocytic vesicles without the formation of toxic membrane pores. The lower pH in endosomes has been shown to be necessary for TAT to escape but the relevance of this pH drop to the biophysical perturbation of the membrane remains to be elucidated (Fischer, Kohler et al. 2004). The interaction of arginine with lipid head groups or with sulfated glycans may make TAT more hydrophobic and further enhance the interaction of TAT with the membrane as has been suggested by

Rothbard et al (Rothbard, Jessop et al. 2004).

Membrane potential has also been suggested to be driving force for the transduction of TAT and other PTDs across the lipid bilayer (Rothbard, Jessop et al.

2004). However, there is scant evidence to support this idea. Membrane depolarizing compounds have an inhibitory effect on transduction, but this effect may be indirect as many cellular processes are disrupted by membrane depolarization, including endocytosis. Disruption of membrane potential may also disrupt the interaction of the transduction domain with the cell surface rather than at the membrane translocation step. TAT has also been shown to cross lipid bilayers in a polarization-dependent manner. However, polylysine peptides cross these bilayers just as effectively as TAT; this is not true in cells, indicating that other factors must be involved (Mitchell, Kim et al. 2000). It has been suggested that the propensity of

guanidino groups to scavenge anions rather than deprotonate, as with lysine, may be an important property responsible for transduction of arginine-rich PTDs (Nishihara,

Perret et al. 2005).

We and others have observed that greater than 90% of the fluorescent signal from labeled peptides or proteins appears in punctate vesicles, not in the nucleus or cytoplasm. Several groups have also shown an improvement in transduction efficiency with endosomolytic agents like chloroquine indicating that much of the peptide remains in vesicles of undetermined fate (Caron, Quenneville et al. 2004; Sloots and Wels 2005). This would appear to be a limitation of transduction if none of this peptide ever enters the cytoplasm. However, this may prove to be a pool that can be exploited to increase the efficiency of transduction. Indeed, Wadia et al showed that a pH-dependent membrane-disrupting peptide fused to TAT could enhance transduction by improving the ability of the peptide to escape from endosomes (Wadia, Stan et al. 2004). Future improvement to transduction efficiency will be attained if we can further exploit this escape bottleneck.

The endocytosis paradigm of TAT also begs many questions with regards to endosome traffic, a subject where very little is currently known. TAT macropinocytotic vesicles are known to traffic along microtubules but the compartments they traffic to or through are unknown (Nakase, Niwa et al. 2004). Macropinosomes traffic along microtubules and are not known to enter the lysosomal pathway. Microscopic evidence indicates that macropinosomes containing TAT move along microtubules but in a back and forth fashion as if their trafficking were impaired (S. Futaki, personal communication).

THERAPEUTIC PROSPECTS

TAT and other protein transduction domains have enormous potential to solve decades-old problems in the delivery of macromolecular therapeutics. The use of information-rich macromolecules has great advantages in specificity and potency that most small molecule therapeutics cannot match. However, traditional gene therapy using viral delivery systems for the introduction of foreign nucleic acids as a means to treat disease is wrought with problems in delivery, serious toxicity and immunogenicity. The use of TAT as delivery vehicle for therapeutic proteins, peptides, antisense and siRNAs avoids many of the drawbacks of gene therapy. We briefly discuss here novel strategies recently devised to enhance the efficiency of transduction, in addition to several applications in particular diseases and cargoes.

Chemical Adjuncts and Modifications

Our enhanced understanding of the membrane interactions of cationic PTDs has paved the way to the introduction of compounds and chemical modifications that enhance uptake of these PTDs. The Matile and Futaki groups have pioneered the use of hydrophobic acidic “activators” for delivery of arginine-rich CPPs (Nishihara,

Perret et al. 2005; Takeuchi, Kosuge et al. 2006). Used in trans as an adjunct, these hydrophobic counteranions appear to enhance uptake by providing improved membrane translocation ability to the PTD, without the toxicity associated with membrane pore-forming agents. Strong binding of the guanidino group to the carboxylate or other anionic moiety on the small molecule leads to strong interaction with hydrophobic membrane components and enhanced cytosolic uptake.

Chemical modifications of the peptide or synthesis of guanidine-rich oligomeric peptide analogs is a growing area in the field of transduction technology.

Peptoids, carbamate polymers, guanidinoglycosides, β-peptides, multivalent or branched peptides and nanoparticles have all been devised to overcome limitations of CPP transduction and enhance the bioavailability or transducibility of the vector

(Wender, Mitchell et al. 2000; Wender, Rothbard et al. 2002; Luedtke, Carmichael et al. 2003). For several years peptides containing D amino acids have been used in the synthesis of TAT and other PTDs to enhance the bioavailability by inhibiting degradation of the peptide by cellular aminopeptidase enzymes. By creating arginine-rich oligomers with a completely synthetic backbone in the form of a β- peptide, peptoid or carbamate chain, enzymatic degradation of the molecule can be reduced for further enhancement of transduction. The use of branched-chain dendrimers or peptides has been shown to increase transduction efficiency and has further demonstrated the principle whereby increased valency and density of TAT and polyarginine enhance uptake (Goun, Pillow et al. 2006; Kawamura, Sung et al. 2006;

Khalil, Kogure et al. 2006). Although multiple barriers exist to the clinical viability of these compounds and modifications, insights and improvements derived from them will likely be critical to the clinical effectiveness of PTD delivery of many peptide, protein and small RNA therapeutics in the near future.

Clinical Applications of TAT and Delivery of Nucleic Acid Cargo

Many groups in widely arrayed fields of clinical and basic biology are working toward TAT-based delivery of therapeutic molecules. The diseases being treated range from cancer, ischemia and neurodegenerative disease and the cargoes range from peptides and full-length proteins to nucleic acids. The TAT-survivin, or

shepherdin, peptide has recently shown promise in the treatment of several cancers including prostate, breast, leukemia, melanoma and glioma (Kim, Woo et al. 2007).

The peptide antagonizes the interaction between Hsp90 and survivin resulting in apoptotic cell death specifically in tumor cells. Another TAT-based strategy for specifically inducing cell death in tumor cells has been the use p53 activating peptides and full-length p53 protein (Michiue, Tomizawa et al. 2005; Snyder, Saenz et al. 2005). These peptides and proteins are effective in treating cancer in mouse models and could possibly be used as an adjunct to more traditional therapies

(Snyder, Meade et al. 2004; Wang and El-Deiry 2004; Michiue, Tomizawa et al.

2005).

Another burgeoning area of research is the use of TAT peptides and proteins in the treatment of ischemic stroke and neurodegenerative disease. TAT conjugated

PKC-δ inhibitor peptides have shown promise in treatment of ischemia-reperfusion diseases in mouse and rat models. These PKC inhibitor peptides have proven to have strong activity in vitro; clinical trials are currently underway to assess the potential of their use to treat human disease (Inagaki, Begley et al. 2005; Bright,

Steinberg et al. 2007). Multiple full-length TAT fusion proteins have also been tested in ischemia and neurodegeneration including Bcl-Xl, glial-derived neurotrophic factor

(GDNF), alpha-synuclein and superoxide dismutase (Kilic, Kilic et al. 2005; Choi, An et al. 2006; Choi, Lee et al. 2006; Kilic, Kilic et al. 2006).

Among the most interesting areas of TAT research is PTD-mediated delivery of siRNAs where a great deal of effort on the part of many researchers has recently met with some success. With the theoretical potential to cure nearly any disease, the

delivery of small RNAs is an extremely hot topic and an important application of transduction technology. PTD mediated delivery of nucleic acids has been an active field for many years as researchers devised delivery systems for DNA plasmids for gene expression and antisense oligos for translational inhibition and splicing modification. Unfortunately, after showing promise in many disease models, the antisense field has stumbled with poor results in clinical trials. The advent of RNAi has reinvigorated TAT-based delivery of nucleic acids because the high potency of siRNAs makes them appear to be perfect cargo for TAT-based delivery. A recent review from our lab highlights many of the strides being made in this prolific area of research (Meade and Dowdy 2007). In addition, the ability of TAT and other PTDs to ferry macromolecular cargo into the cells of live animals has opened up their use in development of diagnosis and imaging technology. A recent and thorough review details these efforts (Bullok, Gammon et al. 2006).

CONCLUDING REMARKS

The human genome, proteomic and structural data available today provide a wealth of data for the derivation of macromolecular therapeutics. However, systemic delivery of macromolecules remains the primary barrier to their use. Bioavailability is of prime importance in the design of any therapeutic and TAT-mediated transduction allows the circumvention of Lipinski’s rule of five and unlocks the potential to use nearly any therapeutic imaginable. The use of TAT as a carrier for therapeutic proteins and peptides has many advantages over traditional gene therapy. The therapeutic is relatively short-lived and lasts only as long as the lifetime of the protein or peptide. Most importantly, non-viral therapy does not require the integration of the nucleic acids into the patients’ genome, with potentially catastrophic consequences.

Additionally, the use of TAT-mediated delivery of macromolecules does not elicit the body’s innate immune response to viral repetitive structures that can both limit the effectiveness of a gene-based therapy and also lead to toxic immunity.

New techniques within the field to assay the uptake of PTDs have paved the way for consistent, quantitative readouts for cytoplasmic translocation that are a vast improvement over the fluorescent peptide microscopy used previously (Loison, Nizard et al. 2005; Aussedat, Sagan et al. 2006). These and other techniques that can distinguish cytoplasmic versus endocytic PTDs are vital for the assessment of transduction efficiency and to understand the mechanism by which these peptides enter cells. Improvements to our ability to assay transduction efficiency will therefore help to further promote the improvement of transduction efficiency. Our ability to more accurately assay transduction efficiency has already led to modifications that increase transducibility of cationic PTDs. Many researchers are making headway in

enhancing delivery of cationic PTDs thereby increasing the bioavailability of general or specific cargo. Together these efforts will pave the way to successful macromolecular therapeutic interventions for multiple human diseases.

GLOSSARY

TAT: the transduction domain derived from the HIV Tat – transactivator of transcription – protein. This short peptide RKKRRQRRR can be linked to multiple large macromolecular cargoes and transport them into cells in vitro and in vivo.

PTD: protein or peptide transduction domain – refers to multiple peptides that, like

TAT, can effect cellular delivery of macromolecules.

CPP: cell penetrating peptide – used interchangeably with PTD, also called permeation peptides.

Macromolecular therapeutics: new field of pharmacology concentrating on the delivery of large bioactive molecules. Differs from gene therapy in that macromolecular therapeutics do not rely on the cellular expression of introduced genetic material.

Bioavailability: the properties of a therapeutic that enable delivery to the site of action. This is a measure of the ability of a therapeutic to enter cells or tissues and also to evade degradation or excretion.

Macropinocytosis: a form of fluid phase endocytosis performed by all cells wherein large circular membrane ruffles fold back on the cell and take up the surrounding medium.

Proteoglycans: sugar-modified proteins ubiquitously expressed on the surface of cells. The surface of all cells is coated with heparan sulfate, chondroitin sulfate and sialic acid modified proteins which are thought to be important for the uptake of TAT.

Polyarginine: a cationic PTD composed of 6-12 arginine residues. The mechanism of TAT and polyarginine transduction appears to be very similar.

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The text of Chapter 2 is a reprint of the material as it appears in

Trends in Molecular Medicine, 2007, Vol 13, No 10, 443-448. Jacob M. Gump and

Steven F. Dowdy. The dissertation author was the primary author of this article.

CHAPTER 3:

REVISED ROLE OF GLYCANS IN TAT-MEDIATED CELLULAR TRANSDUCTION

ABSTRACT

Cellular uptake of the HIV TAT protein transduction domain (PTD) has previously been surmised to occur in a manner dependent on the presence of heparan sulfate proteoglycans expressed ubiquitously on the cell surface. These acidic polysaccharides form a large pool of negative charge that TAT is known to bind avidly and their presence in solution can inhibit transduction. Additionally, sulfated glycans have been hypothesized to aid in the interaction of TAT and other arginine rich peptides with the cell membrane, perhaps aiding their translocation across the membrane. Surprisingly, however, TAT-mediated induction of macropinocytosis and cellular transduction occur in the absence of several acid glycans, including heparan sulfate. Using labeled TAT peptides and fusion proteins, in addition to TAT-Cre recombination-based phenotypic assays, we show that transduction occurs efficiently in glycan deficient CHO mutant cell lines lacking expression of heparan and chondroitin sulfates or sialic acid. Similar results are obtained in cells where glycans have been removed enzymatically. By contrast, enzymatic removal of proteins from the cell surface completely ablates transduction. Our findings support the hypothesis that acidic glycans form a pool of charge that TAT binds on the cell surface, but this binding is independent of the mechanism of transduction or the induction of macropinocytotic uptake by TAT.

INTRODUCTION

Cationic peptide-mediated cellular transduction represents a cell entry modality with enormous potential for the delivery of macromolecular therapeutic agents. The HIV TAT protein basic domain (RKKRRQRRR) and other protein/peptide transduction domains (PTDs) have been used to deliver a wide variety of bioactive cargo into cells in culture and preclinical models in vivo (El-Andaloussi, Holm et al.

2005; Goun, Pillow et al. 2006; Meade and Dowdy 2007). Efficient delivery of peptide, nucleic acid and full-length protein cargoes by cationic PTDs has been demonstrated by many groups and several clinical trials are currently underway using

PTD-mediated delivery. Despite a number of studies on the mechanism used by

PTDs to enter cells, many questions about their uptake remain unanswered (Fischer,

Fotin-Mleczek et al. 2005; Gump and Dowdy 2007; Nakase, Takeuchi et al. 2008;

Heitz, Morris et al. 2009).

We and others have previously shown that TAT PTD and other cationic PTDs induce a ubiquitous form of fluid phase endocytosis termed macropinocytosis and enter cells in this manner (Nakase, Niwa et al. 2004; Wadia, Stan et al. 2004; Kaplan,

Wadia et al. 2005). However, many mechanistic aspects of TAT PTD transduction remain poorly defined. For instance, we have yet to identify the molecules that TAT

PTD binds on the cell surface to induce macropinocytosis, nor do we know how escape from the endosome into the cytoplasm is accomplished. It has previously been shown that TAT PTD avidly binds to sulfated glycans and the presence of acidic polysaccharides like heparin and chondroitin sulfate in solution can compete with TAT

PTD for binding to the cell surface and inhibit transduction (Rusnati, Coltrini et al.

1997; Hakansson, Jacobs et al. 2001; Wadia, Stan et al. 2004). Several studies

55 argue for a significant role for glycans in the transduction of cationic PTDs, with some researchers concluding that transduction is dependent on heparan sulfate (HS) proteoglycans (Tyagi, Rusnati et al. 2001; Silhol, Tyagi et al. 2002; Violini, Sharma et al. 2002; Console, Marty et al. 2003; Richard, Melikov et al. 2005; Nakase, Tadokoro et al. 2007). However, these studies rely predominantly on microscopic visual observations of fluorescently labeled PTD peptides and are therefore not necessarily assaying for the amount of PTD-cargo that has escaped from endocytic vesicles into the cytoplasm. The role of HS proteoglycans in the uptake of PTDs, other cationic molecules and polymers has been explored by several groups and has been reviewed recently (Brooks, Lebleu et al. 2005; Gump and Dowdy 2007; Poon and

Gariepy 2007).

Here, we sought to explore the role of glycans in TAT PTD-mediated transduction using Chinese hamster ovary (CHO) glycan deficient cell lines that are genetically deficient for cell surface expression of heparan and chondroitin sulfate glycosaminoglycans (GAGs) or sialic acids (SA) (Deutscher, Nuwayhid et al. 1984;

Esko, Stewart et al. 1985; Esko, Weinke et al. 1987; Esko 1992). Although previous studies using fluorescently labeled PTD peptides indicated a requisite role for HS in transduction (Tyagi, Rusnati et al. 2001; Console, Marty et al. 2003; Nakase,

Tadokoro et al. 2007), surprisingly, we found via a phenotypic assay that cells lacking heparan sulfate are in fact fully functional for TAT PTD-mediated transduction.

Moreover, transduction occurs efficiently in cells completely lacking all glycosaminoglycans including heparan sulfate, in cells lacking sialic acids and in cells depleted of both together. Our findings support the hypothesis that acidic glycans

56 form a pool of negative charge that TAT PTD binds on the cell surface, but are dispensable for cationic PTD-mediated transduction into cells.

EXPERIMENTAL PROCEDURES

Generation of stable cell lines- Parental CHO-K1 cells and glycosaminoglycan mutant pgsA (745) cells lacking xylosyltransferase necessary for

GAG chain initiation were a gift from Dr. Jeffrey Esko (UCSD). Lec2 cells, which lack activity of the SA golgi transporter SLC35A1 and are extremely deficient in expression of cell surface SA, were a gift from Dr. Ajit Varki (UCSD). Cells were maintained in Ham’s F12 medium (Invitrogen) containing 10% FBS (Sigma). Stable clones were made using a construct (pZ/EG) that contains a Lox-Stop-Lox-EGFP motif resulting in strong EGFP expression only after Cre-mediated recombination.

Cells were transfected with pZ/EG and selected with G418. Multiple subclones were analyzed by flow cytometry for Cre-inducible EGFP expression following transfection with a Cre expression construct and subsequently by treatment with TAT PTD-Cre.

The clones chosen for use in this study had low background EGFP expression and showed comparable induction of EGFP expression after expression of Cre.

Peptide Synthesis and Purification of Recombinant Proteins- Peptides were synthesized via SPPS on a Symphony Quartet Peptide Synthesizer (Rainin) using

Fmoc-protected L amino acids on an amidated support (EMD). Fluorescein labeling was effected by reaction of the peptide with carboxyfluorescein (Sigma) while still on solid phase support. Crude peptides were purified by reverse phase HPLC and confirmed by MALDI-TOF mass spectroscopy.

Recombinant TAT-Cre and Cre proteins were expressed in BL21-

CodonPlus(DE3)-RIPL (Stratagene) using pET28.2 protein expression constructs

(Novagen). Both the TAT PTD-Cre and Cre constructs contain a 6xHis motif at the

58 carboxy-terminus with TAT at the N-terminus in the TAT-Cre expression construct.

Cells were transformed and grown overnight at 37 °C under Kanamycin and

Chloramphenicol selection. Expression was induced with 500 uM IPTG for 4 hours at

37 °C. Bacteria were lysed by sonication in the presence of protease inhibitors, lysozyme, RNAseA and DNAseI. Recombinant Cre was purified from cleared lysate first by Ni-NTA (Qiagen) chromatography followed by ion exchange chromatography using a Mono S column on an AKTA FPLC (GE Healthcare Life Sciences). Purity and concentration of proteins was confirmed by SDS-PAGE gel electrophoresis and

Cre activity was confirmed by in-cell transduction and in-vitro recombination assays.

Recombinant Cre and TAT PTD-Cre were labeled with Alexa-Fluor-546 by reaction with the succinimidyl ester (Invitrogen) for 1 hour under slightly basic conditions according to the company’s protocol. Labeled protein was concentrated and re- purified by ion exchange FPLC and confirmed by gel electrophoresis.

TAT-Cre transduction and recombination assays- Stable CHO-K1, pgsA and

Lec2 LSL-GFP clones were seeded overnight onto 24 well plates at a density of

20,000 cells per well. Cells were washed and incubated in serum-free medium for 1 hour at 37 °C. When pharmacological inhibitors (Sigma) were used, these were added with the serum free medium and added again with recombinant protein. Cells were then treated with Cre / TAT PTD-Cre for 1 hour in serum-free medium at 37 °C or 4 °C, as indicated. Cells were then washed with PBS, trypsinized and replated. At

24 hours after Cre addition, cells were trypsinized and assayed for GFP expression on an LSRII flow cytometer equipped with FACSDiva acquisition and analysis software (BD Biosciences). Live cell population was gated by FSC/SSC and 10,000

59 live cells were used for each analysis. Histograms were generated using FlowJo software (Tree Star).

For trypsin depletion of cell surface proteins, cells were treated with trypsin-

EDTA (Invitrogen) or EDTA-based cell dissociation solution (Sigma) for 15 minutes at

37 °C followed by washes with PBS and 1x soybean trypsin inhibitor (10 mg/mL in

PBS, Sigma). Cells were then treated with TAT PTD-Cre for 1 hour at 37 °C in serum free medium with 1x soybean trypsin inhibitor, followed by trypsinization and replating. Cells were analyzed for recombination at 24 hours as above.

TAT PTD-FITC and labeled TAT PTD-Cre cell association and extracellular binding- CHO-K1, pgsA and Lec2 cells (uncloned or Z/EG LSL-GFP clones were used interchangeably with identical results) were seeded overnight onto 24 well plates at a density of 75,000 cells per well. Cells were washed and incubated in serum-free medium for 1 hour at 37 °C. For cell association, cells were then treated with TAT PTD-FITC or TAT PTD-Cre-Alexa546 for 1 hour in serum-free medium at 37

°C or 4 °C, as indicated. Cells were then placed on ice, washed several times with

PBS, washed with 0.5 mg/mL heparin (Sigma) in PBS, trypsinized and placed on ice for FACS. Cell association was quantitated by FACS as above. For extracellular binding, cells were placed on ice and incubated for 15 minutes with labeled TAT PTD peptide or TAT PTD-Cre in serum-free medium. Cells were then washed several times with ice cold PBS, washed with 0.5 mg/mL heparin in PBS, removed from plate with cell dissociation solution (Sigma) and placed on ice for immediate FACS analysis. For microscopic visualization of cell association, cells were plated on glass coverslips and treated as above with 0.5 µM TAT PTD-Cre-Alexa546 for 1 hour in

60 serum-free medium at 37 °C. Cells were washed several times with PBS, washed with 0.5 mg/mL heparin in PBS and visualized on an Axiovert 200M microscope equipped with a live cell incubation chamber and CCD camera (Zeiss). Images were acquired using Axiovision 4.5 Software (Zeiss) and processed for publication using

Photoshop CS2 (Adobe).

Macropinocytotic Uptake of Neutral Dextran- For flow cytometric analysis

CHO-K1, pgsA and Lec2 cells (uncloned or LSL-GFP clones were used interchangeably with identical results) were seeded overnight onto 24 well plates at a density of 50,000 cells per well. Cells were washed and incubated in serum-free medium for 1 hour at 37°C. Cells were then incubated for 1 hour at 37 °C with 0.5 mg/mL 70 kDa neutral dextran-Texas Red with the indicated concentrations of unlabeled TAT PTD peptide or TAT PTD-Cre protein. Cells were then placed on ice, washed several times with PBS, washed with 0.5 mg/mL heparin (Sigma) in PBS, trypsinized and placed on ice for FACS. Cells were analyzed by FACS as above.

For microscopy, cells were plated on glass coverslips overnight and incubated in serum-free F12 medium for 1 hour at 37 °C. Cells were then incubated for 1 hour at

37 °C with 0.5 mg/mL 70 kDa neutral Dextran-tetramethylrhodamine and 1 µM unlabeled TAT PTD peptide, followed by washes with PBS, 0.5 mg/mL heparin/PBS and microscopy as above.

Heparinase and Sialidase depletion of glycans- CHO-K1, pgsA and Lec2

LSL-GFP cells were seeded overnight on 24 well plates and treated with the indicated concentrations of Heparinase-3 (Sigma) or Sialidase (Neuraminidase from

Arthrobacter ureafaciens, E-Y Laboratories) in serum free medium for 1 hour at 37

°C. After washing with PBS, cells were assayed for TAT PTD-Cre recombination, cell association or dextran uptake, as above. To determine the efficiency of HS depletion, cells were placed on ice and treated with 1 µg/mL biotinylated FGF2 (J. Esko lab) for

15 minutes followed by washes with ice cold PBS, 15 minute incubation with streptavidin-PE-Cy5 (BD Biosciences) and washes with PBS. Following removal from the plate with cell dissociation solution, cells were analyzed by FACS. To assay SA depletion, cells were incubated with FITC-conjugated wheat germ agluttinin (Sigma) on ice for 15 minutes, followed by PBS washing and FACS.

RESULTS

Fluorescently labeled TAT PTD actively associates with cells lacking glycans-

To obtain a quantitative measure of transduction efficiency and to reconcile our preliminary data with those of previous researchers, we measured, via flow cytometry, the cell association of fluorescently-labeled TAT PTD peptide and TAT PTD-Cre in wild-type and glycan deficient CHO cells. Cells treated with fluoresceinated TAT PTD peptide (TAT PTD-FITC) exhibit differences in cell association, with less TAT peptide association in the glycan deficient cells, particularly at 5 µM where there was a 30%-

40% reduction in cell association (Figure 3.1A & 3.1B). Incubation of cells at 4 °C is known to inhibit macropinocytotic uptake of TAT PTDs (Nakase, Niwa et al. 2004;

Wadia, Stan et al. 2004; Kaplan, Wadia et al. 2005). We detected differences between wild type CHO cells and HS and SA deficient cells in cell surface binding of

TAT PTD peptides at 4 °C (Figure 3.1C) that correlate with differences in TAT PTD-

FITC cell association, indicating a reduction in extracellular binding capacity in glycan deficient cells. We also examined uptake differences of a TAT PTD fusion protein, namely TAT PTD-Cre (37 kDa). Consistent with the TAT PTD peptide results above, cells lacking HS and SA also had reduced TAT PTD-Cre-Alexa546 cell association relative to wild type glycan positive CHO cells (Figure 3.2A). Cells treated with TAT

PTD-Cre-Alexa546 also exhibit punctate macropinocytotic staining as visualized by live cell microscopy (Figure 3.2B). Extracellular binding of labeled TAT PTD-Cre at 4

°C (Figure 3.2C) revealed similar differences in the inability of the glycan-deficient cells to bind TAT PTD-Cre and TAT PTD-FITC, further indicating that the differences seen in cell association may be due to differential extracellular binding capacities rather than a defect in transduction per se.

TAT PTD-Cre transduction and recombination occur efficiently in CHO glycan mutant cell lines- Analysis of cell association and uptake of fluorescently labeled PTD peptides and fusion proteins may be incomplete and potentially misleading. To avoid these pitfalls, we assayed HS and SA deficient cells for induction of a cellular phenotype that is dependent on PTD-cargo escape into the cytoplasm/nucleus.

Previously we devised a TAT PTD-Cre – loxP-STOP-loxP (LSL) GFP reporter system as a readout for TAT PTD-Cre transduction. Entrance of TAT PTD-Cre into the nucleus results in recombination and elimination of the LSL-GFP transcriptional terminator, resulting in expression of GFP. Unlike visual assays employing fluorescently labeled proteins or peptides that may inadvertently measure cell surface binding and endocytotic uptake (in the absence of endosomal escape) TAT PTD-Cre- induced recombination and GFP expression is an unequivocal measure of transduction (Wadia, Stan et al. 2004). Because of its stringency, this system may underestimate the efficiency of the transduction phenomena being observed, yet it gives an unparalleled phenotypic measure of transduction with minimal potential for false positives or misinterpretation.

We analyzed TAT PTD-Cre transduction in LSL-GFP stable clones of parental glycan-positive wild-type CHO-K1 and derivative glycan deficient cell lines pgsA and Lec2. Treatment of each of these cell lines with TAT PTD-Cre induced

GFP expression in a dose-dependent manner as observed by flow cytometry (Figure

3.3A & 3.3C) and microscopy (Figure 3.3B). This demonstrates the ability of TAT

PTD-Cre to enter cells and transit to the nucleus to recombine the LSL genetic element in the absence of HS and SA. Although cells lacking glycans showed a slight shift of the dose-response curve, both HS and SA deficient cells reached the

64 same maximal response at 2 µM. Similar results were obtained using recombinant

8xArg PTD-Cre fusion protein (data not shown). In contrast, control recombinant Cre protein (no PTD) showed no induction of GFP above background levels (Figure

3.3A). Inhibition of macropinocytosis by Cytochalasin-D resulted in a dramatic reduction in TAT PTD-Cre mediated recombination and GFP induction (Figure 3.3A &

3.3B).

TAT PTD-mediated transduction occurs via macropinocytosis in wild type and glycan-deficient cells- We measured the effect of several macropinocytotic chemical inhibitors on TAT PTD-Cre transduction. Wild type, HS-deficient and SA- deficient CHO cells were treated with three different macropinocytotic inhibitors:

Amiloride (a proton pump inhibitor that specifically affects macropinocytosis) (Figure

3.4A), Wortmannin (a kinase inhibitor) (Figure 3.4B), and Cytochalasin-D (an inhibitor of F-actin polymerization) (Figure 3.4C). Treatment with all three macropinocytotic inhibitors resulted in dramatic reductions of TAT PTD-Cre transduction into cells as measured by a dose-dependent decrease in TAT PTD-Cre mediated recombination of the LSL-GFP reporter in the absence of significant cellular toxicity (Figure 3.4A-C, inset). In addition, enzymatic removal of cell surface proteins by trypsin also potently inhibited TAT PTD-Cre-mediated transduction into cells (Figure 3.4D).

We have previously shown that TAT PTDs stimulate their own cellular uptake by increasing the basal rate of macropinocytosis (Wadia, Stan et al. 2004; Kaplan,

Wadia et al. 2005). Treatment of wild type, HS-deficient and SA-deficient CHO cells with a known macropinocytosis marker, fluorophore-labeled 70 kDa neutral dextran, in combination with unlabeled TAT PTD peptide resulted in a dose-dependent

65 increase in dextran uptake in response to TAT PTD in all three cell types (Figure

3.5A). Consistent with this observation, microscopic visualization also showed the presence of labeled dextran in TAT PTD-induced macropinosomes in all three cell types (Figure 3.5B), suggesting that TAT PTD stimulation of macropinocytosis occurs independently of both HS and SA. Furthermore, enzymatic depletion of HS and SA with Heparinase and Sialidase has no effect on dextran uptake indicating that cells lacking both HS and SA in combination are capable of TAT PTD-induced macropinocytosis (Figures 3.5C & 3.5D).

Enzymatic removal of glycans has a negligible effect on transduction- In order to confirm our assays using genetically glycan deficient cell lines, we sought to determine if the same results would be obtained by enzymatic depletion of HS and

SA. Our observations with mutant CHO cell lines could be due to compensation for a deficiency that is only evident after acute depletion, or a strong phenotype might only be evident in cells lacking HS and SA in combination. Efficient removal of cell surface

HS and SA was accomplished using heparinase and sialidase enzymes (Figures

3.6E & 3.6F). Pre-treatment of cells with these enzymes has little to no effect on TAT-

Cre transduction and recombination efficiency (Figures 3.6A & 3.6B). Likewise, there is no substantial effect on TAT-FITC peptide cell association (Figures 3.6C & 3.6D).

In contrast, binding of fibroblast growth factor 2 (FGF2) and wheat germ agglutinin

(WGA), ligands known to bind specifically to HS and SA, respectively, were greatly reduced in the treated cells (Figures 3.6E & 3.6F). Taken together, these observations demonstrate that cationic TAT PTD-mediated cellular delivery of a Cre recombinase cargo and induction of macropinocytosis occur independent of HS and

SA.

DISCUSSION

Based primarily on microscopic visualization, previous studies concluded that cells lacking proteoglycans are refractory to cellular transduction by several PTDs, including the TAT PTD (Tyagi, Rusnati et al. 2001; Sandgren, Cheng et al. 2002;

Console, Marty et al. 2003; Elson-Schwab, Garner et al. 2007; Nakase, Tadokoro et al. 2007; Nascimento, Hayashi et al. 2007), while other researchers obtained conflicting results (Silhol, Tyagi et al. 2002; Violini, Sharma et al. 2002; Console,

Marty et al. 2003). We originally intended to use HS-deficient mutants as transduction null cells to facilitate our research into the mechanism of transduction.

Our original hypothesis was that a HS proteoglycan served as a receptor for the TAT

PTD and was responsible for stimulating macropinocytotic uptake. However, to our surprise, experiments using a cellular phenotypic assay, not merely visualization, demonstrate that cells lacking HS or SA are fully competent to efficiently transduce

TAT PTD-Cre into cells.

To further explore our unexpected preliminary findings, we first looked at cell association of fluorescent TAT PTD-FITC peptide and TAT PTD-Cre-Alexa546 in wild-type and glycan deficient cells. Our data corroborate the work of previous researchers indicating a reduction in cell association of labeled TAT PTD peptide and labeled TAT PTD-Cre protein in glycan deficient cell lines (Console, Marty et al. 2003;

Nakase, Tadokoro et al. 2007). However, the differences we see are not so dramatic as to warrant the conclusion that transduction is not occurring in these cells: the cell association of TAT PTD-FITC and TAT PTD-Cre-Alexa546 are well above control in both HS and SA deficient cells. Nevertheless, there are clear and consistent differences in the magnitude of transduction we see in glycan-deficient cells that likely

67 explain the conclusions of previous authors that transduction does not occur in cells lacking HS. The differences we see in cell association in cells lacking glycans correlate well with differences in extracellular binding of TAT PTD peptide and TAT

PTD-Cre protein. This indicates that the minor differences seen in cell association are due to reduced extracellular TAT-PTD binding capacity of glycan-deficient cells, rather than a deficiency in transduction pathways.

To further explore transduction in the absence of glycans, we sought to generate a transduction data set based on a phenotypic assay rather than using cell association of labeled TAT as an approximation of transduction. To accomplish this, we generated stable Cre-responsive cell lines from CHO-K1, pgsA and Lec2 cells using a loxP-Stop-loxP-GFP reporter gene. As we have shown previously, cell lines with an integrated LSL-GFP construct, do not express GFP in the absence of Cre recombinase. However, after treatment of LSL-GFP cells with exogenous recombinant TAT PTD-Cre protein, GFP expression is induced. Using this system, we can determine the relative efficiency of TAT PTD transduction without the caveats of visualization based on labeled peptides and proteins. Assays that rely on fluorescent tags to measure cellular uptake cannot discriminate between endosomal and cytosolic fractions of the cell and may grossly overestimate the actual fraction of peptide and cargo that has transduced into the cell (escaped from the endosome).

We prefer to refer to these measures as “cell association” because they do not sufficiently distinguish cells that have fluorescent label in endocytic vesicles or bound to the cell surface, from cells where the peptide and cargo have actually escaped endosomes into the cytoplasm. This distinction is important because endosomal escape is requisite for transduction; the majority of labeled TAT PTD taken up by cells

68 is present in endosomes and most researchers would agree that only a small proportion escapes to the cytoplasm. In addition, for the purposes of most therapeutic interventions, the inside of the endosome is essentially the outside of the cell; no biological response will be elicited simply by uptake into vesicles. The importance of escape is further illustrated by the increase in transduction efficiency seen with endosome disrupting peptides (Wadia, Stan et al. 2004; El-Sayed, Futaki et al. 2009).

Treatment of Cre-responsive cells with TAT PTD-Cre revealed a trivial difference between wild type and HS or SA deficient cells. If transduction were not occurring in these cells, we would expect to see a reduction to baseline as seen with recombinant Cre lacking TAT PTD or treatment with cytochalasin-D. The efficient manner with which TAT PTD-Cre enters glycan-deficient cells is strong and convincing evidence that transduction occurs in the absence of extracellular glycosaminoglycans and sialic acids.

Further evidence that transduction is occurring in these cells is the existence of TAT PTD-induced macropinocytotic uptake of 70 kDa neutral dextran in glycan deficient pgsA and Lec2 cells. Were these cells refractory to transduction, or if there were another pathway for entry, we would not expect to see dextran uptake increased in response to TAT PTD. Furthermore, the mode of uptake is macropinocytosis in wild type and glycan-deficient cell lines, as evidenced by the similar reductions in transduction efficiency seen after treatment with chemical inhibitors of macropinocytosis.

We show here, in an extensive exploration, that TAT PTD-mediated transduction does not require heparan sulfate or sialic acids, it occurs efficiently in their absence. By our very stringent Cre-loxP system and by more conventional fluorescent visualization and cytometry, TAT PTD enters cells lacking HS or SA and cells that have been depleted of both in combination. Furthermore, TAT PTD- induced macropinocytotic fluid-phase uptake is intact in glycan deficient cells indicating that TAT PTD does not require these glycans to stimulate endocytosis. By contrast, depletion of cell surface proteins with protease results in drastically reduced transduction efficiency, indicating that a cell surface protein is necessary for TAT PTD transduction (though not necessarily as a receptor). These data suggest that cationic peptides like TAT PTD may induce macropinocytotic uptake and transduction via binding to a protein on the cell surface rather than through interactions with glycans or direct interaction with the membrane.

Together, our findings are consistent with the hypothesis that the dense forest of extracellular glycans forms a pool of negative charge that TAT PTD binds on the cell surface. Differences in this charge pool affect the efficiency of TAT transduction to varying degrees, but are independent of the ability of PTDs to transduce cells or to induce macropinocytotic uptake. Further study to determine the extracellular proteins required for transduction will offer important insights into the mechanism of transduction of TAT PTD as well as other PTDs.

FIGURES

A B CHO-K1 pgsA CHO-K1 100 300 300 pgsA

e Lec2 c # 80 # n 200 200 e c s C e l C e l e r 60 100 100 o u l M a x i m u ) F

o f 40 n 0 0 a 0 1 2 3 0 1 2 3 e ( % 10 10 10 10 10 10 10 10 M 20 FITC FITC

0 Lec2 0 0.1 0.5 1.0 2.5 5.0 5.0 300 Untreated µM TAT-FITC 0.1 µM 0.5 µM 37 °C 37 °C 4 °C # 200 1.0 µM 2.5 µM C e l 100 5.0 µM T A - F I C C 5.0 µM 4 °C CHO-K1 0 100 0 1 2 3 e pgsA 10 10 10 10 c ) n

m Lec2 FITC e 80 u c s m i e r x o

a 60 u l M F f o

n 40 a % e (

M 20

0 0 0.1 0.5 2.5 5.0 5.0 µM µM TAT-FITC +Trypsin 4 °C - No Heparin Wash

FIGURE 3.1 TAT peptide cell association and cell surface binding in glycan- deficient cells. A, TAT peptide cell association in CHO-K1 parental and glycan mutant derivative cell lines pgsA and Lec2. Cells were treated with the indicated concentrations of fluoresceinated TAT peptide (TAT-FITC) at 37 °C for 1 hour followed by washes with PBS, heparin and trypsin to remove extracellular peptide. Cells were placed on ice and immediately analyzed by flow cytometry. B, Representative FACS profiles for the experiment in A. C, TAT peptide cell surface binding at 4 °C. Cells were treated for 15 minutes with TAT-FITC peptide followed by washes with PBS, non-enzymatic cell dissocation and flow cytometry.

A C CHO-K1 CHO-K1 100 100 pgsA pgsA e e c c Lec2 80 Lec2 n n 80 e e c c s s e e 60 r r 60 o o u u l l M a x i m u ) M a x i m u ) F F 40 o f o f 40 n n a a e e ( % ( % M M 20 20

0 0 0 0.1 0.25 0.5 1.0 2.5 2.5 0 0.1 0.25 1.0 2.5 2.5 µM +Trypsin µM TAT-Cre-Alexa546 µM TAT-Cre-Alexa546 37 °C 4 °C 4 °C - No Heparin Wash B CHO-K1 pgsA Lec2 - C r e T A T A l e x a 5 4 6

FIGURE 3.2 TAT fusion protein cell association and cell surface binding in glycan-deficient cells. A, Flow cytometric analysis of cells treated for 1 hour at 37 °C at the indicated concentrations of recombinant TAT-Cre labeled with Alexa Fluor 546. Cells were washed thoroughly with PBS, heparin and trypsin to remove extracellular TAT fusion protein. B, Live cell photomicrographs of the indicated cell lines treated with 1 uM TAT-Cre- Alexa546 for 1 hour at 37 °C. TAT-Cre treatment was followed by extensive washing with PBS and heparin. The scale bar indicates 5 µm. C, TAT-Cre-Alexa546 cell surface binding at 4 °C. Cells were treated for 15 minutes with TAT-Cre-Alexa546 followed by PBS wash, non- enzymatic cell dissociation and flow cytometry.

A B 100 1 K - O

80 H C s l l CHO-K1 e 60 C pgsA TAT-Cre A +

Lec2 s P g F

CHO-K1 p G 40

% pgsA Cre Lec2 20 2 c e L 0 0 0.25 0.5 1.0 1.5 2.0 2.0 µM 0 0.25 0.5 1.0 2.0 µM Cre + CytoD µM TAT-Cre + CytoD C CHO-K1 pgsA Lec2 400 400 400 Untreated 0.25 µM 300 300 300 e #

0.5 µM r C 1.0 µM - T

C e l 200 200 200 2.0 µM A T 100 100 100 2.0 µM + CytoD

0 0 1 2 3 0 0 1 2 3 0 0 1 2 3 10 10 10 10 10 10 10 10 10 10 10 10 GFP GFP GFP

FIGURE 3.3 TAT-Cre transduction occurs in the absence of glycosaminoglycans and sialic acids in CHO parental and glycan-deficient cell lines. A, TAT-Cre recombination in CHO-K1 and glycan mutant derivative pgsA and Lec2 cells with a stably integrated Lox-Stop-Lox-EGFP expression construct (pZ/EG). Cells were treated with purified recombinant TAT-Cre fusion protein or recombinant Cre without TAT at the indicated concentrations for 1 hour followed by trypsinization and replating. Flow cytometry was performed at 24 hours after Cre addition. As indicated, control cells were treated with CytochalasinD to inhibit macropinocytosis for 1 hour at 37 °C immediately preceding and during TAT-Cre treatment. B, Photomicrographs of TAT-Cre recombination- induced EGFP expression in parental and glycan mutant cells with stably integrated pZ/EG construct. C, FACS profiles of representative samples from the experiment in A.

A mM Amiloride B µM Wortmannin s

0 .1 .25 .5 1 s l l 0 .25 .5 1 2 l 100 l 100 e e C 100 C 100 e e l 80 l b b 80 a 80 a i 60 i 80 60 V V s s l l l

40 l %

% 40 e e C 60 C 60 +

+ CHO-K1 P P

F pgsA 40 F

G 40 G Lec2 % CHO-K1 % 20 pgsA 20 Lec2 0 0 - 0 0.1 0.25 0.5 1.0 mM Amiloride - 0 0.1 0.25 0.5 1.0 µM Wortmannin 1 µM TAT-Cre 1 µM TAT-Cre

µM CytochalasinD

C s D 0 .1 .25 .5 1 l 100 l e 100 No Treatment 100 C e l

80 b Trypsin 30 Minutes a

80 i

60 V 80 CDS 30 Minutes s l l s 40 % l e l e C 60 60 C

+ CHO-K1 + P pgsA P F

40 F G Lec2 40 G % % 20 20

0 0 - 0 0.1 0.25 0.5 1.0 µM CytochalasinD 0 0.5 2.5 TAT-Cre µM 1 µM TAT-Cre

FIGURE 3.4 Macropinocytotic inhibitors and TAT-Cre transduction in glycan- deficient cells. A, CHO-K1, pgsA and Lec2 cells with a stably-integrated Lox-Stop-Lox- EGFP (pZ/EG) construct were treated with the indicated concentrations of amiloride, an inhibitor of macropinocytosis, for 1 hour. Cells were then treated with TAT-Cre protein in the presence of amiloride for 1 hour followed by trypsinization and replating. EGFP expression was assayed by flow cytometry at 24 hours after TAT-Cre addition. Inset: cell viability as assayed by flow cytometry immediately following TAT-Cre treatment. B, Treatment with wortmannin, a kinase inhibitor as in A. C, CytochalasinD, an inhibitor of F-actin and macropinocytosis as above. D, Depletion of cell-surface proteins by trypsin. CHO-K1 Z/EG cells were treated with trypsin or cell dissociation solution for 30 minutes, followed by washes with PBS and trypsin inhibitor and treatment with TAT-Cre for 1 hour. Cells were then washed, trypsinized and replated. EGFP expression was assayed by FACS at 24 hours following TAT-Cre treatment.

C A 160 CHO-K1

200 CHO-K1 e e pgsA c c n

n pgsA ) 180 140 e

l Lec2 e c o c s r 160 Lec2 s t e e n r

r 120

o 140 o o C o n t r l ) u C u l l

f 120 F o f F

o 100

100 n n a % ( % a ( e e 80 80 M M 60 40 60 Dextran 0.5 2.0 Dextran Dextran Dextran Only + TAT + TAT µM TAT + Heparinase B D CHO-K1 pgsA Lec2 160 CHO-K1

e pgsA c n

l ) 140 e Lec2 c k D s e r 7 0 120 o C o n t r u l D e x t r a n - T M R F o f 100 n a ( % e 80 M

60 Dextran Dextran Dextran + TAT + TAT + Sialidase

FIGURE 3.5 TAT-induced macropinocytotic fluid-phase uptake is intact in glycan-deficient cells. A, CHO parental and glycan mutant cells were treated with unlabeled TAT peptide or TAT-Cre in the presence of Texas Red labeled 70 kDa neutral dextran for 1 hour at 37 °C. After washing and trypsinization, cells were assayed for dextran uptake by FACS. B, Live cell photomicrographs of the indicated cell lines following treatment with 1 µM TAT in the presence of 70 kDa dextran-tetramethylrhodamine. C, TAT-induced uptake of 70 kDa dextran-Texas Red following treatment with 200 mU heparinase for 1 hour at 37 °C; TAT concentration 1 µM. D, Cells were treated with 200 mU sialidase followed by dextran and TAT as in C.

B A TAT-Cre CHO-K1 TAT-Cre CHO-K1 80 pgsA pgsA

s Lec2 80 Lec2 l s l l l e

60 e C

C 60 + + P

40 P

F 40 F G G

% 20 % 20

0 0 0 50 200 200 0 50 200 200 no no mU Heparinase TAT-Cre mU Sialidase TAT-Cre C D TAT-Pep-FITC CHO-K1 TAT-Pep-FITC CHO-K1

e pgsA pgsA e c c

n Lec2 Lec2 n

e 100 e 100 c c s s e e r 75

r 75 o o u u l 50 l F 50 F M a x i m u ) n M a x i m u ) n a

25 a 25 e ( % e ( % M 0 M 0 0 50 200 200 0 50 200 200 mU Heparinase no TAT mU Sialidase no TAT

E CHO-K1 F CHO-K1

e FGF2 WGA e c pgsA pgsA c n ) ) n

e Lec2 Lec2 s t s e c i 30 t 16 i c s n n s e r U e U

r 12 o

y 20 o u y r l r u a l

F 8 a r t r F i t n 10 i b n a b

r 4 r a e A A e ( ( M 0 0 0 50 200 200 M 0 50 200 200 no FGF2 no WGA mU Heparinase mU Sialidase

FIGURE 3.6 Enzymatic depletion of heparan sulfate and sialic acids does not impair TAT transduction in parental or glycan-deficient cells despite efficient removal of glycans. A & B, The indicated Lox-Stop-Lox-EGFP stable cell lines were treated for 1 hour with heparinase or sialidase as indicated, at 37 °C, followed by 1 µM TAT- Cre for 1 hour at 37 °C, trypsinization and replating. EGFP expression was assayed by flow cytometry 24 hours after TAT-Cre treatment. C & D, The indicated cell lines were treated with heparinase and sialidase enzymes as in A & B, followed by treatment with 1 µM TAT-FITC peptide for 1 hour at 37 °C, immediately followed by PBS, heparin and trypsin washes and flow cytometry. E & F, heparinase and sialidase treatment, as above, results in substantial reductions in cell-surface binding of heparan sulfate-specific FGF2 and sialic acid-specific WGA, respectively.

REFERENCES

Brooks, H., B. Lebleu and E. Vives (2005). "Tat peptide-mediated cellular delivery: back to basics." Adv Drug Deliv Rev 57(4): 559-77.

Deutscher, S. L., N. Nuwayhid, P. Stanley, E. I. Briles and C. B. Hirschberg (1984). "Translocation across Golgi vesicle membranes: a CHO glycosylation mutant deficient in CMP-sialic acid transport." Cell 39(2 Pt 1): 295-9.

El-Andaloussi, S., T. Holm and U. Langel (2005). "Cell-penetrating peptides: mechanisms and applications." Curr Pharm Des 11(28): 3597-611.

El-Sayed, A., S. Futaki and H. Harashima (2009). "Delivery of macromolecules using arginine-rich cell-penetrating peptides: ways to overcome endosomal entrapment." AAPS J 11(1): 13-22.

Elson-Schwab, L., O. B. Garner, M. Schuksz, B. E. Crawford, J. D. Esko and Y. Tor (2007). "Guanidinylated neomycin delivers large, bioactive cargo into cells through a heparan sulfate-dependent pathway." J Biol Chem 282(18): 13585- 91.

Esko, J. D. (1992). "Animal cell mutants defective in heparan sulfate polymerization." Adv Exp Med Biol 313: 97-106.

Esko, J. D., T. E. Stewart and W. H. Taylor (1985). "Animal cell mutants defective in glycosaminoglycan biosynthesis." Proc Natl Acad Sci U S A 82(10): 3197- 201.

Esko, J. D., J. L. Weinke, W. H. Taylor, G. Ekborg, L. Roden, G. Anantharamaiah and A. Gawish (1987). "Inhibition of chondroitin and heparan sulfate biosynthesis in Chinese hamster ovary cell mutants defective in galactosyltransferase I." J Biol Chem 262(25): 12189-95.

Fischer, R., M. Fotin-Mleczek, H. Hufnagel and R. Brock (2005). "Break on through to the other side-biophysics and cell biology shed light on cell-penetrating peptides." Chembiochem 6(12): 2126-42.

Goun, E. A., T. H. Pillow, L. R. Jones, J. B. Rothbard and P. A. Wender (2006). "Molecular transporters: synthesis of oligoguanidinium transporters and their

application to drug delivery and real-time imaging." Chembiochem 7(10): 1497-515.

Gump, J. M. and S. F. Dowdy (2007). "TAT transduction: the molecular mechanism and therapeutic prospects." Trends Mol Med 13(10): 443-8.

Hakansson, S., A. Jacobs and M. Caffrey (2001). "Heparin binding by the HIV-1 tat protein transduction domain." Protein Sci 10(10): 2138-9.

Heitz, F., M. C. Morris and G. Divita (2009). "Twenty years of cell-penetrating peptides: from molecular mechanisms to therapeutics." Br J Pharmacol.

Kaplan, I. M., J. S. Wadia and S. F. Dowdy (2005). "Cationic TAT peptide transduction domain enters cells by macropinocytosis." J Control Release 102(1): 247-53.

Meade, B. R. and S. F. Dowdy (2007). "Exogenous siRNA delivery using peptide transduction domains/cell penetrating peptides." Adv Drug Deliv Rev 59(2-3): 134-40.

Nakase, I., M. Niwa, et al. (2004). "Cellular uptake of arginine-rich peptides: roles for macropinocytosis and actin rearrangement." Mol Ther 10(6): 1011-22.

Nakase, I., T. Takeuchi, G. Tanaka and S. Futaki (2008). "Methodological and cellular aspects that govern the internalization mechanisms of arginine-rich cell- penetrating peptides." Adv Drug Deliv Rev 60(4-5): 598-607.

Nascimento, F. D., M. A. Hayashi, A. Kerkis, V. Oliveira, E. B. Oliveira, G. Radis- Baptista, H. B. Nader, T. Yamane, I. L. Tersariol and I. Kerkis (2007). "Crotamine mediates gene delivery into cells through the binding to heparan sulfate proteoglycans." J Biol Chem 282(29): 21349-60.

Poon, G. M. and J. Gariepy (2007). "Cell-surface proteoglycans as molecular portals for cationic peptide and polymer entry into cells." Biochem Soc Trans 35(Pt 4): 788-93.

Sandgren, S., F. Cheng and M. Belting (2002). "Nuclear targeting of macromolecular polyanions by an HIV-Tat derived peptide. Role for cell-surface proteoglycans." J Biol Chem 277(41): 38877-83.

Tyagi, M., M. Rusnati, M. Presta and M. Giacca (2001). "Internalization of HIV-1 tat requires cell surface heparan sulfate proteoglycans." J Biol Chem 276(5): 3254-61.

Wadia, J. S., R. V. Stan and S. F. Dowdy (2004). "Transducible TAT-HA fusogenic peptide enhances escape of TAT-fusion proteins after lipid raft macropinocytosis." Nat Med 10(3): 310-5.

The material presented in Chapter 3, in part, has been submitted for publication. Jacob M. Gump and Steven F. Dowdy. The dissertation author was the primary investigator and author of this material.

CHAPTER 4:

TAT PROTEIN AND PEPTIDE TRANSDUCTION OCCURS BY ACTIVATING RAC1-

DEPENDENT MEMBRANE RUFFLING AND RAB5 & RAB34-MEDIATED

MACROPINOCYTOSIS

ABSTRACT

Cellular uptake of TAT protein transduction domain (PTD; or cell penetrating peptide (CPP)) and other cationic PTDs, has been shown occur via actin and lipid raft-dependent macropinocytosis. However, the gene products necessary for TAT

PTD transduction have not been fully elucidated, and surprisingly little is known about macropinocytosis, a process that nearly all cells are thought to perform. We show that TAT PTD induces actin and Rac1-dependent membrane ruffling, leading to macropinocytosis; while RNAi depletion of Rac1 leads to a substantial inhibition of

TAT PTD-mediated transduction. Using live cell microscopy we see that TAT-induced membrane ruffles co-localize with Rab34 and labeled TAT PTD colocalizes with Rab5 endocytic vesicles. In addition, TAT PTD transduction and macropinocytotic uptake are altered by expression of GTP binding mutants of the GTPases Rab5 and Rab34, whose roles in macropinocytosis have recently been indicated. Our data further support the role of macropinocytosis in uptake of cationic PTDs and implicate key players in macropinocytosis as necessary for PTD-mediated transduction.

INTRODUCTION

The therapeutic delivery of bioactive macromolecules by protein transduction domains (PTDs) has the potential to create a new paradigm in medicine by expanding the range of pharmaceuticals well beyond typical small molecule drugs.

The HIV Tat protein basic domain – TAT (RKKRRQRRR) and other PTDs have been used to efficiently deliver a wide variety of cargo into cells in culture, stem cells and preclinical models in vivo (El-Andaloussi, Holm et al. 2005; Goun, Pillow et al. 2006;

Meade and Dowdy 2007; Eguchi, Meade et al. 2009). Efficient delivery of peptide, nucleic acid and large protein cargoes by PTDs has been demonstrated by many groups and clinical trials are underway using PTD-mediated delivery. Despite a number of studies on the mechanism used by PTDs to enter cells, many questions about their uptake remain unanswered (Fischer, Fotin-Mleczek et al. 2005; Gump and

Dowdy 2007; Nakase, Takeuchi et al. 2008; Heitz, Morris et al. 2009).

We and others have previously shown that TAT PTD and other cationic PTDs induce macropinocytosis, a form of fluid-phase endocytosis performed by all cell types, and enter cells in this manner (Nakase, Niwa et al. 2004; Wadia, Stan et al.

2004). The molecular players involved in TAT PTD-mediated macropinocytotic uptake remain relatively undefined, with actin and Rac1 being the only gene products shown to be necessary for cationic PTD uptake. While much more is known about macropinocytosis in general, it still remains perhaps the most loosely-defined form of endocytosis (Kerr and Teasdale 2009). Macropinocytosis shares many molecular and phenotypic characteristics with phagocytosis, but rather than being limited to specialized cells, it is carried out by most cells (Conner and Schmid 2003).

Macropinocytosis is characterized by the induction of actin polymerization-mediated

82 apical or peripheral membrane ruffles that form circular invaginations, or cups; these membrane extrusions then fuse to form macropinosomes (Swanson 2008; Kerr and

Teasdale 2009). The processes of actin-dependent membrane ruffling and macropinocytosis are induced by growth factor stimulation of cells and are known to depend on the activities of the small GTPases Rac1, cdc42, and Arf6, as well as phosphoinositide 3-kinase (PI3K), p21-activated kinase (PAK1)(Dharmawardhane,

Schurmann et al. 2000; Schafer, D'Souza-Schorey et al. 2000; West, Prescott et al.

2000; Liberali, Kakkonen et al. 2008). While there is strong evidence that TAT PTD uptake occurs via macropinocytosis, with the exception of Rac1, the roles for these proteins in the cellular transduction of cationic PTDs have not been fully explored.

The GTPase Rab5 has been shown by several groups to play a role in membrane ruffling and macropinocytosis. Rab5 is required for proper control of endocytosis, and growth factor receptor-induced activation of membrane ruffling and endocytosis are known to be dependent on its activity (Barbieri, Roberts et al. 2000;

Zerial and McBride 2001; Palamidessi, Frittoli et al. 2008). The small GTPase Rab34 has been implicated in macropinocytotic uptake, where it was found to colocalize with membrane ruffles, nascent macropinosomes and a subset of Rab5-positive endosomes (Sun, Yamamoto et al. 2003). More recently, Rab5 and Rab34 were shown to participate in the macropinocytotic entry of coxsackievirus (Coyne, Shen et al. 2007).

We show here that TAT PTD-mediated macropinocytotic uptake proceeds through both Rab5 and Rab34 compartments. Disruption of Rab5 and Rab34 endocytic pathways via overexpression of GTP binding mutants, alters

83 macropinocytotic uptake of TAT. We also show that TAT PTD transduction depends on activation of Rac and concomitant membrane ruffling, which leads directly to macropinocytotic uptake. Our results confirm that TAT PTD-mediated uptake proceeds via Rac1, Rab5 and Rab34-dependent macropinocytosis.

EXPERIMENTAL PROCEDURES

Generation of stable cell lines- Cos7 cells were a gift from the laboratory of

Dr. Marilyn Farquhar and were maintained in DMEM (Invitrogen) with 10% FBS

(Sigma). Wild-type CHO-K1 cells were a gift from the laboratory of Dr. Jeffrey Esko

(UCSD), these cells were maintained in Ham’s F12 medium (Invitrogen) containing

10% FBS. H1299 cells were maintained in DMEM with 10% FBS. Stable Cre- responsive clones (H1299-LSL-GFP CHO-LSL-GFP, or CHO-LSL-Cherry) were generated using expression constructs with either a LoxP-Stop-LoxP-EGFP or LoxP-

Stop-LoxP-mCherry motifs that allow expression of the fluorescent protein only after

Cre-mediated recombination. H1299-LSL-GFP and CHO-LSL-GFP cells were made via transfection of pZ/EG (Novak, Guo et al. 2000) and selection with G418

(Invitrogen). The LSL-mCherry plasmid was constructed by inserting, via PCR-based cloning, mCherry into a CMV-LoxP-Stop-LoxP construct derived from the

Tex.LoxP.EGFP cell line (Lin, Jo et al. 2004). Multiple subclones for each line were analyzed by flow cytometry for Cre-inducible fluorescent protein expression following transfection with a Cre expression construct and subsequently by treatment with TAT-

Cre. The clones chosen for use in this study had low background fluorescence and showed a two log induction of fluorescence after expression of Cre, as assayed by flow cytometry.

Peptide Synthesis and Purification of Recombinant Proteins- Peptides were synthesized via SPPS on a Symphony Quartet Peptide Synthesizer (Rainin) using

Fmoc-protected L amino acids on an amidated support (EMD).

Tetramethylrhodamine (TMR) labeling was effected by reaction of the peptide with carboxytetramethylrhodamine (Sigma) while on solid phase support. Crude peptides

85 were purified by reverse phase HPLC and confirmed by MALDI-TOF mass spectroscopy.

Recombinant TAT-Cre and Cre proteins were expressed in BL21-

CodonPlus(DE3)-RIPL (Stratagene) using pET28.2 protein expression constructs

(Novagen). Both the TAT-Cre and Cre constructs contain a 6xHis motif at the carboxy-terminus with TAT at the N-terminus in the TAT-Cre expression construct.

Cells were transformed and grown overnight at 37 °C under Kanamycin and

Chloramphenicol selection. Expression was induced with 500 uM IPTG for 4 hours at

Cre and TAT-Cre were labeled with Alexa-Fluor-594 by reaction with the succinimidyl ester (Invitrogen) for 1 hour under slightly basic conditions according to the company’s protocol. Labeled protein was concentrated and re-purified by ion exchange FPLC and confirmed by gel electrophoresis.

TAT-Cre transduction and recombination assays- Stable H1299-LSL-GFP,

CHO-LSL-GFP and CHO-LSL-mCherry clones were seeded overnight onto 24 well plates at a density of 20,000 cells per well. Cells were washed and incubated in serum-free medium for 1 hour at 37 °C. When cytochalasin-D (Sigma) was used to inhibit transduction, it was added with the serum-free medium and added again along

86 with recombinant protein. Cells were treated with TAT-Cre for 1 hour in serum-free medium at 37 °C or 4 °C, as indicated. Cells were then washed with PBS, trypsinized and replated. At 24 hours after Cre addition, cells were trypsinized and assayed for

EGFP or mCherry expression on an LSRII flow cytometer equipped with FACSDiva acquisition and analysis software (BD Biosciences). Live cell population was gated by FSC/SSC and 10,000 transfected (GFP or mCherry-positive) cells were used for each analysis.

Rab constructs and transfections- GFP-Rab5 (WT, Q79L and S34N) and

GFP-Rab4 (WT, Q67L and N121I) constructs were obtained as N-terminal EGFP fusions and were a gift from Dr. Robert Lodge (University of Toronto). GFP-Rab34

(WT, Q111L and T66N) N-terminal fusions were a gift from the laboratory of Dr. Mel

Silverman (University of Toronto). mCherry-Rab5 fusions were constructed by PCR- based cloning of mCherry in place of EGFP into the above constructs. All plasmids were verified by sequencing.

For transient transfections, H1299-LSL-GFP, CHO-LSL-GFP or CHO-LSL- mCherry cells were plated overnight in 6 well plates to sub-confluency. Cells were then transfected using FuGENE 6 (Roche) for 4 hours, followed by replating into 24 well plates at the optimal density for either labeled TAT cell association assays or

TAT-Cre transduction assays. Alternately, cells were replated onto glass coverslips at low density for microscopy, as described below.

TAT-TMR and labeled TAT-Cre-Alexa-594 cell association- CHO-K1 cells were seeded overnight onto 24 well plates at a density of 75,000 cells per well. Cells

87 were washed and incubated in serum-free medium for 1 hour at 37 °C. For cell association, cells were then treated with TAT-TMR or TAT-Cre-Alexa-594 for 1 hour in serum-free medium at 37 °C or 4 °C, as indicated. Cells were then placed on ice, washed several times with PBS, washed with 0.5 mg/mL heparin (Sigma) in PBS, trypsinized and placed on ice for FACS. Cell association was quantitated by FACS as above.

Microscopy- For live cell microscopic visualization of colocalization, transfected cells were plated on glass coverslips and treated with either 0.25 µM

TAT-Cre-Alexa-594 or .5 µM TAT-TMR for 1 hour in serum-free medium at 37 °C.

Cells were washed several times with PBS, washed with 0.5 mg/mL heparin in PBS and visualized on an Axiovert 200M microscope equipped with a live cell incubation chamber and CCD camera (Zeiss). Images were acquired using Axiovision 4.5

Software (Zeiss) and processed for publication using ImageJ (NIH) and Photoshop

(Adobe).

Transferrin Uptake and Macropinocytotic Uptake of Neutral Dextran- For flow cytometric analysis of dextran fluid-phase uptake, GFP-Rab transfected CHO cells were seeded overnight onto 24 well plates at a density of 50,000 cells per well. Cells were washed and incubated in serum-free medium for 1 hour at 37°C. Cells were then incubated for 1 hour at 37 °C with 0.5 mg/mL 70 kDa neutral Dextran-

Rhodamine with 1 µM unlabeled TAT peptide. Cells were then placed on ice, washed several times with PBS, washed with 0.5 mg/mL heparin (Sigma) in PBS, trypsinized and analyzed by FACS, gating on GFP positive cells, as above. For microscopy of dextran colocalization, Rab-transfected cells were plated on glass coverslips

88 overnight and incubated in serum-free F12 medium for 1 hour at 37 °C. Cells were then incubated for 1 hour at 37 °C with 0.5 mg/mL 70 kDa neutral dextran-Texas Red or dextran-Cascade Blue (Invitrogen) and 1 µM unlabeled TAT peptide, followed by washes with PBS, 0.5 mg/mL heparin/PBS and microscopic visualization as above.

For flow cytometric analysis of transferrin uptake, GFP-Rab transfected CHO cells were seeded overnight onto 24 well plates at a density of 50,000 cells per well.

Cells were washed and then incubated for 15 minutes at 37 °C with 0.1 mg/mL

Transferrin-Texas Red (Invitrogen). Cells were then placed on ice, washed several times with PBS, trypsinized and analyzed by FACS, gating on GFP positive cells, as above. For microscopy of transferrin colocalization, Rab-transfected cells were plated on glass coverslips overnight. Cells were then incubated for 30 minutes at 37 °C with

0.1 mg/mL Transferrin-Texas Red, followed by washes with PBS and microscopic visualization as above.

TAT induction of membrane ruffling and actin reorganization- For fixed-cell phalloidin assays, Cos7 cells were plated on glass slides overnight at low density.

Cells were then incubated in media without serum or antibiotics for 1 hour at 37 °C, followed by treatment with PMA (Sigma) or unlabeled TAT peptide at 37 °C for the indicated times. Cells were immediately placed on ice, fixed with 4% paraformaldehyde and stained with phalloidin-rhodamine (Sigma) for 30 min. and

Hoechst 33342 (Invitrogen) for 5 min. After mounting, cells were visualized as above.

For live cell YFP-actin experiments, Cos7 cells were plated overnight in 6 well plates at ~80% confluency. Cells were then transfected with YFP-actin using

FuGENE 6 (Roche) for 4 hours, cells were then trypsinized and replated on glass cover slips and allowed to sit down for 18-24 hours. After serum starving the cells for

1-2 hours, cells were treated with TAT peptide or PMA at the indicated concentrations while recording time-lapse microscopy.

Rac activity and Rac1a RNA interference- CHO cells were plated overnight at a density of 200,000 cells per well in a 6 well plate. Cells were then treated with

PDGF (Invitrogen) or unlabeled TAT peptide at 37 °C for the indicated times. Cells were immediately placed on ice and Rac activity was determined using an ELISA- based Rac GTP pull-down kit (Cytoskeleton, Denver, CO) exactly per the kit instructions.

For RNAi depletion of Rac1, H1299-LSL-GFP cells were plated overnight to sub-confluency and transfected with control and Rac1 siRNA pool (Ambion) using

Lipofectamine2000 (Invitrogen). After a 4 hour transfection, cells were replated and at

48 hours after transfection, either treated with 1 µM TAT-Cre to assay for transduction efficiency as above or lysed and subjected to immunoblotting with anti-Rac1

(Upstate) or anti-γ-Tubulin (Sigma) antibodies.

RESULTS

TAT PTD stimulates Rac activation and actin-dependent membrane ruffling-

It has previously been shown that uptake of polyarginine and MPG PTDs occurs in a

Rac1-dependent manner (Gerbal-Chaloin, Gondeau et al. 2007; Nakase, Tadokoro et al. 2007). We found that depletion of Rac1 by RNAi caused a dramatic decrease in the efficiency of TAT-Cre transduction in H1299-LSL-GFP cells (Figure 4.1A).

Treatment of H1299 cells with unlabeled TAT PTD peptide resulted in a significant transient increase in Rac activity similar to that seen with PDGF treatment, which is known to activate Rac (Figure 4.1B). This increase in Rac activity results in actin cytoskeletal rearrangements and the formation of peripheral and circular membrane ruffles (Swanson 2008; Kerr and Teasdale 2009). Cos7 cells treated with unlabeled

TAT or PMA showed a marked change in actin dynamics, with TAT inducing both peripheral and apical membrane ruffling as visualized by phalloidin staining (Figure

4.2A). Time-lapse microscopy of live cells treated with unlabeled TAT peptide reveals dynamic induction of peripheral actin ruffling and the de novo formation of macropinosomes (Figure 4.2B and 4.2C).

Rab34 is recruited to TAT PTD-induced membrane ruffles and overexpression of GTP-bound Rab34 enhances transduction and macropinocytosis-

Cells transfected with GFP-Rab34 followed by treatment with TAT or PMA show enhanced colocalization of GFP-Rab34 and actin (Figure 4.3A). Interestingly, when we overexpressed GTP-bound GFP-Rab34 (Q111L) we found that it resulted in an enhancement of cellular transduction of TAT-Cre and increased colocalization of labeled TAT-Cre-Alexa-594 with Rab34-positive endosomes (Figures 4.3B and 4.3C).

The enhancement of transduction appears to be due to an increase in

91 macropinocytosis, as uptake of TMR-labeled 70 kDa neutral dextran was also enhanced by expression of the Rab34 Q111L mutant (Figure 4.3D). In addition, GFP-

Rab34 colocalizes with a marker for macropinocytosis, 70 kDa neutral dextran-Texas

Red in cells treated with 1 µM TAT (Figure 4.3E).

Rab5 is associated with Rab34-positive TAT-induced macropinosomes and colocalizes with TAT-containing macropinocytotic vesicles- After transfecting cells with GFP-Rab34 and mCherry-Rab5, we treated cells with unlabeled TAT peptide in the presence of Cascade Blue-labeled neutral dextran. In these cells, Dextran Blue- containing endocytic vesicles were surrounded by both GFP-Rab34 and mCherry-

Rab5 (Figure 4.4A) indicating that TAT macropinosomes are associated, at least transiently, with both Rab5 and Rab34. Ectopic expression of Rab5 Q79L is known to result in phenotypically aberrant, large endosomes (Stenmark, Parton et al. 1994;

Stenmark, Valencia et al. 1994). After treatment of transfected cells with fluorescently labeled TAT-TMR peptide or TAT-Cre-Alexa-594 protein (data not shown), GFP-Rab5

Q79L vesicles contained endocytosed TAT PTD (Figure 4.4B). This may indicate that upon entering cells, TAT PTD transits through a Rab5 compartment. In addition, transient transfection of GFP-Rab5 Q79L resulted in diminished cell association of labeled TAT-TMR (Figure 4.4C) and TAT-Cre-Alexa-594 (data not shown), as assayed by flow cytometry.

TAT-Cre transduction and recombination are altered by Rab5-Q79L expression- We analyzed TAT PTD-Cre transduction in CHO-LSL-GFP and CHO-

LSL-Cherry stable clones transiently expressing GFP-Rab fusion proteins. Cells expressing mCherry-Rab5 Q79L showed a modest reduction in recombination

92 indicating a decreased transduction efficiency compared to control cells (Figure

4.4D). By contrast, expression of Rab4 (Figure 4.4E) and Rab11 (data not shown) proteins had no effect on TAT-Cre transduction and recombination. It should be noted that the decrease in transduction seen with Rab5 Q79L was independent of the fluorescent tag (mCherry or GFP) used (Figures 4.4D and 4.4E).

Overexpression of Rab5-Q79L results in decreased macropinocytotic fluid- phase uptake- We showed previously that TAT PTDs trigger their own cellular uptake by stimulating macropinocytosis (Figures 4.1 & 4.2) (Wadia, Stan et al. 2004; Kaplan,

Wadia et al. 2005). Transiently expressed GFP-Rab5-Q79L endosomes contain 70 kDa neutral dextran-Texas Red, a marker for fluid-phase macropinocytotic uptake

(Figure 4.5A). Treatment of CHO cells transiently expressing GFP-Rab5 Q79L with unlabeled TAT peptide in the presence of 70 kDa neutral dextran-TMR results in decreased uptake of dextran (Figure 4.5B). Likewise, treatment of cells with cytochalasin-D, an inhibitor of F-actin and macropinocytosis results in decreased uptake of neutral dextran (Figure 4.5B). By contrast, transferrin-Texas Red colocalizes with GFP-Rab5 Q79L (Figure 4.5C), but expression of Rab5 Q79L does not alter transferrin uptake dynamics (Figure 4.5D).

DISCUSSION

Recent evidence supports the macropinocytic model for TAT PTD uptake and suggests that Rac1, a key effector of membrane ruffling and macropinocytosis is necessary for PTD-mediated transduction (Gerbal-Chaloin, Gondeau et al. 2007;

Nakase, Tadokoro et al. 2007). To further understand the role of Rac activity in TAT transduction we depleted Rac1 using siRNA and saw a substantial reduction in TAT-

Cre transduction efficiency, indicating that the activity of Rac is necessary for transduction. Transfection of Rac1 dominant-negative (T17N) results in a similar reduction in TAT-Cre recombination (data not shown). To further understand TAT’s ability to induce macropinocytosis we looked at Rac activity in response to treatment of cells with TAT peptide. TAT peptide is known to induce macropinocytosis, however the mechanism responsible for this activity is unclear. Treatment of cells with TAT peptide induces Rac GTPase activity to an extent similar to that seen with PDGF treatment. This induction of Rac activity corresponds well with a substantial increase in membrane ruffling and formation of macropinosomes, following treatment of cells with TAT. Together, these data suggest that TAT induces Rac1 dependent membrane ruffing and macropinocytosis which mediate its uptake into cells.

Recent research has shown the empirical evidence for globalization of corporate innovation is very limited. And as a corollary, the market for technologies is shrinking. As a world leader, it is important for America to provide systematic research grants for our scientists. I believe there will always be a need for us to have a well-articulated innovation policy with emphasis on human resource development

(Phillips, Armstrong et al. 2003).

Rab34 is a small GTPase which has been suggested to have a role in macropinocytosis (Sun, Yamamoto et al. 2003; Sun and Endo 2005; Coyne, Shen et al. 2007). While only a handful of publications have been published on Rab34, an array of roles have been suggested for it: Rab34 interacts with golgi proteins (Colucci,

Campana et al. 2005; Speight and Silverman 2005), has been shown to regulate lysosome positioning (Wang and Hong 2002), has roles in macropinocytosis (Sun,

Yamamoto et al. 2003; Coyne, Shen et al. 2007) and exocytosis Goldenberg

(Goldenberg, Grinstein et al. 2007) and is implicated in the entry of several pathogens, including E. coli, coxsackievirus, herpesvirus and mycobacterium (Coyne,

Shen et al. 2007; Gutierrez, Mishra et al. 2008; Laakkonen, Makela et al. 2009;

Raghu, Sharma-Walia et al. 2009). In our hands, Rab34 appears to play a role in TAT

PTD transduction. GFP-Rab34 colocalizes with actin at membrane ruffles induced by

TAT and expression of constitutive-active Rab34 Q111L leads to a substantial increase in macropinocytotic uptake of neutral dextran. In addition, endocytic vesicles containing labeled TAT colocalize with GFP-Rab34 Q111L and overexpression of the GTP-locked Q111L mutant leads to a modest but significant increase in transduction efficiency, as assayed by TAT-Cre recombination. These data suggest that TAT PTD uptake into macropinosomes is mediated by Rab34 and that increased Rab34 activity leads to an increase in macropinocytosis and TAT transduction. An alternate interpretation of our data is that Rab34 is involved in trafficking of macropinosomes and that the over-expression of Rab34 Q111L pushes macropinosomes down a pathway that enhances uptake and reduces recycling back to the cell surface. However, previous research supports the former conclusion that

Rab34 activity leads to increased macropinocytosis (Sun, Yamamoto et al. 2003).

Rab5 is a small GTPase with multiple roles in endocytosis and endosomal dynamics (Zerial and McBride 2001). Evidence for the role of Rab5 in macropinocytosis is multi-fold: Rab5 is activated by receptor tyrosine tyrosine kinases and its activity is necessary for the formation of Ras-induced circular membrane ruffles (Lanzetti, Palamidessi et al. 2004; Schnatwinkel, Christoforidis et al. 2004),

Rac1 is a Rab5 effector (Palamidessi, Frittoli et al. 2008) and inhibition of Rab5 activity results in reductions in macropinocytotic uptake (Coyne, Shen et al. 2007).

We found that TAT transduction also depends on Rab5 although its role, surprisingly, appears to be inhibitory. TAT-induced dextran-containing endosomes colocalize with both Rab34 and Rab5 Q79L, indicating at least a transient macropinosome compartment shared by both Rab5 and Rab34. This compartment is not evident in cells expressing either wild-type Rab5 or S34N mutant; cells expressing Rab5 Q79L show pronounced colocalization of TAT-TMR PTD peptide, TAT-Cre-Alexa594 and

70 kDa neutral dextran, which is not seen in with Rab5 WT or Rab5 S34N. We were also surprised to find that overexpression of Rab5 Q79L was not excitatory toward transduction, but inhibitory. This finding was supported by the fact that overexpression of Rab5 Q79L also inhibited uptake of 70 kDa neutral dextran. By microscopy, the uptake and cell association of TAT-TMR, TAT-Cre-Alexa594, 70 kDa neutral dextran and transferrin all appear to be enhanced by overexpression of Rab5

Q79L. However, quantitative FACS analysis shows that this is not the case: Rab5

Q79L inhibits uptake of TAT peptide, TAT-Cre and neutral dextran, and has no effect on transferrin uptake. That constitutive-active Rab5 Q79L inhibits transduction and macropinocytosis is difficult to reconcile with the work of previous researchers, whose findings are themselves contradictory (Bucci, Parton et al. 1992; Ceresa, Lotscher et al. 2001; Lanzetti, Palamidessi et al. 2004). Nevertheless, our work here supports a

96 dynamic role for Rab5 in the macropinocytotic uptake of TAT-PTD: TAT peptide and

TAT-Cre fusion protein are both present in Rab5 Q79L endosomes and expression of this GTPase-deficient mutant inhibits both TAT-induced macropinocytosis and recombination by TAT-Cre. The reduction in transduction efficiency and reduced cell association of both TAT PTD and neutral dextran may indicate that altered trafficking to large, Rab5 Q79L-positive endosomes leads to the endo-lysosomal destruction pathway or, alternately, a recycling pathway to the cell surface.

We have shown previously that TAT PTD-mediated transduction occurs by activation of lipid raft-dependent macropinocytosis, independent of dynamin, clathrin and caveolin (Wadia, Stan et al. 2004; Kaplan, Wadia et al. 2005). Here, we show that TAT induces Rac1-activation and actin polymerization-dependent membrane ruffling and subsequent macropinocytosis. Rab34 is also shown to be associated with actin at sites of membrane ruffling and with TAT-induced macropinosomes. In addition, expression of Rab34 constitutive-active mutant results in enhanced TAT transduction by increasing macropinocytosis, while Rab5 constitutive-active mutant inhibits transduction by an unknown mechanism. Further research on small GTPases known to be involved in macropinocytosis like Arf6, Ras, CDC42 and RhoG, in addition to the endocytic kinases Src, Pak1, PI3K and PKCα/ε, and PLCγ will greatly enhance our knowledge of prototypical macropinocytosis and its relationship to PTD- induced macropinocytic uptake and cellular transduction.

FIGURES

A 70 B 8

60 7 Control Rac1 6 50 αRac1 s l l

e 5 C

40 αTubulin C o n t r l + A c t i v y P 4 F O v e r G 30

R a c 3 % F o l d 20 2

1 10 0 0 PDGF TAT TAT TAT Control Rac1 No treatment 0.1 µM 1 µM 1 µM 1 µM siRNA 2 min 1 min 2 min 5 min

FIGURE 4.1 TAT PTD activates Rac1 and TAT transduction is Rac1-dependent. A, TAT-Cre recombination in H1299-LSL-GFP cells transfected with control or Rac1 siRNA pool. Inset, depletion of Rac1 compared to γ-tubulin control by western blotting. B, Rac activity in H1299 cells treated with PDGF or TAT peptide for the indicated times.

A No treatment PMA B No treatment PMA 5 min 0.1 µM 5 min 3 min 0.1 µM 3 min YFP-Actin

Rhod- Phalloidin

20 µm 10 µm TAT TAT TAT TAT 1 µM 5 min 1 µM 10 min 1 µM 3 min 1 µM 3 min C

YFP-Actin

FIGURE 4.2 TAT PTD induces Rac1-dependent membrane ruffling and macropinocytosis. A, Cos7 cells were treated with PMA or TAT peptide for the indicated times, then fixed and stained with phalloidin-rhodamine to visualize actin-mediated membrane ruffling. B, Live cell microscopy of Cos7 cells expressing YFP-Actin treated with TAT peptide or PMA at the indicated concentrations. Snapshots taken at 3 minutes post-treatment reveal induction of actin-mediated membrane ruffling. Arrows indicate nascent macropinosomes formed at sites of intense ruffling events. C, Time-lapse series from the bottom left panel of B shows induction of ruffling followed by the formation of macropinocytic cups and macropinosomes, as indicated by the arrows.

no treatment 0.1 µM PMA 5 min A B GFP 70 Rab34 WT Rab34 Q111L 60 Rab34 T66N

40 C e l s

20 % m C h e r y +

Hoechst 10 Phalloidin GFP-Rab34 0 1 µM TAT 5 min 1 µM TAT 10 min no treatment 1 µM TAT-Cre 1 µM TAT-Cre + CytoD

C D Merge GFP-Rab34 TAT-Cre-594 7500 GFP Rab34 Q111L Rab34 WT Rab34 T66N

W T 7000

6500 R a b 3 4

F l u o r e s c n 6000 U n i t s

Q 1 L 5500

A r b i t a y 5000 R a b 3 4 D e x t r a n - T M R 4500 T 6 N M e a n 4000 R a b 3 4 3500 Dextran Only Dextran + 1 µM TAT E GFP-Rab34 Dextran-Texas Red

FIGURE 4.3 GFP-Rab34 localizes to TAT-induced membrane ruffles and macropinosomes, and Rab34 Q111L expression alters TAT transduction and macropinocytosis. A, Cos7 cells were transfected with GFP-Rab34 and treated with TAT or PMA for the indicated times followed by fixation and staining with phalloidin. B, CHO-LSL- mCherry cells were transfected with the indicated GFP-Rab34 constructs. At 18 hours, cells were treated with 1 uM TAT-Cre for 1 hour at 37 °C, trypsinized and replated. Flow cytometry of transfected (GFP+) cells reveals the percentage of Rab34-expressing cells where TAT-Cre recombination has occurred. C, CHO cells were transfected with the indicated GFP-Rab34 constructs. Cells were then treated with Alexa-594-labeled TAT-Cre for 1 hour at 37 °C followed by washes with PBS and heparin. D, CHO cells transfected with GFP-Rab34 were incubated with 70 kDa neutral dextran-tetramethylrhodamine in the presence or absence of 1 µM unlabeled TAT peptide. E, Cos7 cells were transfected with GFP-Rab34 WT followed by treatment with 70 kDa neutral dextran-tetramethylrhodamine in the presence of 1 µM unlabeled TAT peptide.

100

GFP-Rab34 mCherry-Rab5 Q79L Dextran-Cascade Blue

D B Merge GFP-Rab5 TAT-TMR 80 mCherry 70 mCh-Rab5 WT W T mCh-Rab5 Q79L 60 mCh-Rab5 S34N R a b 5 50 C e l s 40 Q 7 9 L

% G F P + 30 R a b 5 20

10 S 3 4 N 0

R a b 5 no Cre 0.25 0.5 1 2 2 µM + CytoD µM TAT-Cre C 3500 GFP E 70 GFP-Rab5 WT

e 3000 GFP-Rab5 Q79L 60 c n

e GFP-Rab5 S34N c

s 50 e

r 2500 s o C e l s t i u l n F 40 U y R

r 2000 a M r t

i 30 b r % m C h e r y + A T A - 1500

n 20 a e M 1000 10

0 500 Mock GFP WT QL SN WT QL SN no treatment 1 µM TAT-TMR GFP-Rab4 GFP-Rab5 No Cre 1 µM TAT-Cre

FIGURE 4.4 TAT-PTD traffics through Rab5 endosomes and TAT transduction is altered in cells expressing Rab5 Q79L. A, Cos7 cells were transfected with both GFP-Rab34 and mCherry-Rab5 Q79L and incubated with 70 kDa dextran-cascade blue in the presence of 1µM TAT PTD peptide. B, CHO cells expressing the indicated GFP-Rab5 constructs were treated with TAT-TMR peptide for 30 minutes, followed by washes with PBS and heparin, and fluorescence microscopy. C, CHO cells expressing the indicated GFP-Rab5 constructs were treated with TAT-TMR peptide for 1 hour, followed by washes with PBS and heparin, and flow cytometry. D, FACS analysis of recombination in CHO-LSL-GFP cells expressing the indicated mCherry-Rab5 constructs following treatment with TAT-Cre. E, FACS analysis of recombination in CHO-LSL-mCherry cells expressing the indicated GFP-Rab constructs. Cells were first transfected with the indicated GFP-Rab plasmids, replated overnight, treated with TAT-Cre and trypsinized for FACS 24 hours later.

101

FIGURE 4.5 Rab5 Q79L expression impairs TAT induction of macropinocytosis with no effect on transferrin uptake. A, CHO cells expressing the indicated GFP-Rab5 constructs were treated for 1 hour with 1 µM unlabeled TAT peptide in the presence of 70 kDa dextran-Texas Red, followed by PBS washes and microscopy. B, CHO cells expressing the indicated GFP-Rab5 constructs were treated for 1 hour with 1 µM unlabeled TAT peptide in the presence of 70 kDa dextran-TMR, followed by PBS washes and FACS. C, Live cell photomicrographs of cells treated with transferrin-Texas Red after transfection with the indicated GFP-Rab5 constructs. D, FACS analysis of GFP-Rab5-expressing cells following treatment with transferrin-Texas Red.

102

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The material presented in Chapter 4 is being prepared for publication. Jacob

M. Gump and Steven F. Dowdy. The dissertation author was the primary investigator and author of this material.

CHAPTER 5:

DISCUSSION

Peptide transduction remains one of the few viable means to deliver large molecule therapeutics clinically. However, the clinical success of medicinal macromolecules is hampered by the issue of bioavailability. While peptide transduction domains confer on cargo the ability to enter cells both in vivo and in vitro, the greatest barrier to their widespread clinical use is still poor bioavailability of the transduction domain and cargo. Aside from clearance from the bloodstream in vivo, there are two bottlenecks at the cellular level which reduce the effective concentration and effectiveness of PTD-mediated delivery. One major restriction is undoubtedly the uptake of PTDs into cells and yet another significant restriction lays therein, the release from endocytic vesicles to the cytoplasm. Understanding the bases for these two connected processes will undoubtedly lead to clinically translatable advancements in transduction technology (El-Andaloussi, Holm et al.

2005; Wadia and Dowdy 2005; Tunnemann, Martin et al. 2006; Gump and Dowdy

2007; Heitz, Morris et al. 2009).

The finding that TAT peptide transduction occurs via macropinocytosis has led to a wholesale change in the way peptide transduction is viewed mechanistically.

The knowledge that the membrane translocation step takes place in endocytic vesicles rather than at the cell surface creates new opportunities for enhancements to transduction efficiency and several advances in the uptake of TAT and other PTDs have been made based on the endocytic paradigm (Wadia, Stan et al. 2004; Perret,

Nishihara et al. 2005; Takeuchi, Kosuge et al. 2006). We and many others have

107

108 observed that the vast majority of labeled PTD taken up by cells is present, and presumably trapped, in endocytic vesicles. Based on what is known about macropinocytosis, and other forms of endocytosis as well, if there is no specific receptor present in endosomes to bind and sort cargo for recycling or retrograde transport, then the cargo will eventually be trafficked down the lysosomal degradation pathway. While some anecdotal evidence suggests that PTD-containing macropinosomes have altered trafficking, this has not been proved. Further study comparing growth factor-induced macropinosomes and their trafficking to TAT macropinosomes would shed some light on this subject.

The hypothesis that most of the PTD in macropinocytic vesicles is trapped in a dead end pathway leading to degradation is supported by the observations that chemical, mechanical and peptide-induced endosomal disruption all lead to increases in transduction efficiency (Caron, Quenneville et al. 2004; Fischer, Kohler et al. 2004;

Wadia, Stan et al. 2004; Takeuchi, Kosuge et al. 2006; Kobayashi, Nakase et al.

2009). In addition, the timing of PTD induction of ruffling, macropinocytosis and TAT-

Cre recombination all point to the very fast trafficking of TAT macropinosomes down the endo-lysosomal pathway (Nakase, Tadokoro et al. 2007). TAT induces Rac- dependent ruffling leading to macropinocytosis and Rac GTPase activity reaches a maximum at around 1 minute after TAT addition; likewise, TAT-stimulated Rac- dependent ruffling begins around 1 minute and subsides by 5 minutes after TAT addition. This correlates nicely with PCR data from our lab indicating that TAT-Cre recombination is evident within minutes and reaches a plateau by 30 minutes after treatment. Together, these data point to very quick uptake into and release of TAT from nascent macropinosomes, indicating that whatever is present in endocytic

109 vesicles (early or late endosomes) may be permanently trapped (El-Sayed, Futaki et al. 2009). If this were not the case, we would expect to see slow release of TAT-Cre from vesicles and therefore slow kinetics of recombination. The use of pH-dependent endosome-disrupting fusion peptides has allowed for increased release of peptide and cargo from macropinosomes and the use of peptide-specific membrane disrupting agents has allowed for similar transduction enhancements (Wadia, Stan et al. 2004; Michiue, Tomizawa et al. 2005; Perret, Nishihara et al. 2005; Takeuchi,

Kosuge et al. 2006; El-Sayed, Futaki et al. 2009). Other tactics used to generate enhanced transduction efficiency are UV-inducible membrane disruption and the use of sound waves, which have been used to burst liposomes and have been suggested as a method to increase endosomal release (Matsushita, Noguchi et al. 2004).

My data have helped to advance our understanding of TAT transduction in two realms: first, the mechanistic contribution of glycans in transduction, and second, the induction, uptake and trafficking of TAT by macropinosomes. First, I have found that the uptake and transduction of TAT occurs independent of the presence of glycosaminoglycans and sialic acids on the cell surface which likely excludes a role for proteoglycans in both the induction of macropinocytosis and in membrane translocation as has been suggested (Nakase, Tadokoro et al. 2007). Multiple other authors have concluded that transduction does not occur without heparan sulfate proteoglycans (Tyagi, Rusnati et al. 2001; Console, Marty et al. 2003; Argyris,

Kulkosky et al. 2004; Elson-Schwab, Garner et al. 2007; Gerbal-Chaloin, Gondeau et al. 2007; Poon and Gariepy 2007). It is difficult to resolve these apparently disparate findings, but the findings of others were, by-and-large, based on visualization and when I personally examine their data, in many cases I do not reach the same

110 conclusions as the authors. In addition, with regards to our TAT-Cre results, I find it impossible to explain how we might have obtained the results we did if transduction were not occurring. There is only one explanation for a positive result using TAT-Cre: the protein entered the cell and the nucleus (in an active state) where it recombined the LoxP-stop-LoxP motif and turned on GFP expression. Together with the finding that protease treatment impairs transduction, our data strongly support a glycan- independent mechanism for TAT induction of macropinocytosis and escape from endosomes. Glycosaminoglycans are nevertheless responsible for uptake to some extent as they appear to enhance the ability of TAT to bind cells and thereby increase transduction efficiency.

The second area of my thesis work delineates a role for Rabs in the endocytosis and trafficking of PTDs which has not been previously explored.

Furthermore, the Rabs involved in regulation and trafficking of other macropinocytotic cargoes have only been loosely defined (Sun, Yamamoto et al. 2003; Lanzetti,

Palamidessi et al. 2004; Coyne, Shen et al. 2007). I have explored the roles of Rab4,

Rab5, Rab7, Rab11 and Rab34 in TAT macropinocytosis. Of these, Rabs 5, 7, and

34 appear to be involved in TAT transduction. The part played by Rab7 is not clear and requires further elucidation. Rabs 5 and 34 are clearly associated with TAT macropinosomes and appear to affect transduction but in somewhat unexpected ways. For instance, expression of constitutive active Rab5 (and to a certain extent wild-type) appears to diminish transduction efficiency, yet, TAT is found preferentially associated with Rab5 constitutive active endosomes. These findings were initially difficult to rationalize, but they are consistent with the hypothesis that Rab5 promotes the trafficking TAT macropinosomes to an endosomal compartment not conducive to

111 vesicular escape. Alternatively, Rab5 Q79L may be inhibiting uptake by modulating macropinocytosis. This supposition is supported by Rab5’s ability to modulate Rac activity and by my data showing decreased macropinocytotic uptake upon expression of Rab5 constitutive active mutants (Barbieri, Roberts et al. 2000; Lanzetti,

Palamidessi et al. 2004; Palamidessi, Frittoli et al. 2008).

The function of Rab34 in PTD uptake appears to be more straightforward. I have found that expression of Rab34 constitutive active mutant enhances transduction. This finding is augmented by the observation that Rab34 is associated with TAT-induced membrane ruffles and that Rab34 is associated with TAT-induced macropinosomes and labeled TAT-containing vesicles (this colocalization is enhanced in the presence of Rab5 Q79L). The role I have found with regard to Rab34 is consistent with the hypothesis that Rab34 is a macropinocytotic Rab and previous reports that Rab34 is localized to ruffles and macropinosomes (Chen, Han et al.

2003; Sun, Yamamoto et al. 2003; Coyne, Shen et al. 2007; Raghu, Sharma-Walia et al. 2009). However, previous research indicating that dominant negative Rab34 inhibits macropinocytosis is not supported by my data. Instead, I have found that constitutive active Rab34 enhances macropinocytosis which is, nevertheless, consistent with the same model. Further clarification of the roles of Rabs 5 and 34, in addition to Rab7, by depleting these proteins with RNAi (or genetically) will contribute to our understanding of their part in TAT PTD transduction and macropinocytosis.

The data contained in this dissertation further support the integral role macropinocytosis plays in PTD uptake and enhance our understanding of the molecules TAT uses to bind cells, induce macropinocytosis and escape from

112 endosomes. Based on my findings, the supposition that glycans, particularly heparan sulfate proteoglycans, are responsible for transduction needs modification. Other phenotypic and quantitative assays for transduction using multiple PTDs will clarify our knowledge in this realm. In addition, our knowledge with regards to trafficking of

TAT macropinosomes is beginning to be understood, based on my research.

Extending my findings will be crucial to understanding when, where and how TAT escapes from macropinocytic vesicles. One major gap in our understanding is how growth factor induced macropinocytosis differs from TAT macropinocytosis, if at all.

Mechanistic comparisons of these two pathways will help to better define prototypical macropinocytosis at the molecular level while enhancing our understanding of potentially clinically important steps in TAT PTD-mediated cellular transduction.

Furthermore, a stepwise and methodical observation of the intracellular fate of TAT and the compartments with which it associates will help us understand and perhaps manipulate trafficking to our advantage.

PTD-mediated cellular transduction has given us the ability to deliver macromolecules for research and therapeutic use, but our deficient understanding of the mechanism responsible for transduction hinders our ability to enhance and apply this technology more effectively. We and others have used PTDs to deliver multiple large cargoes for laboratory and therapeutic purposes (Gupta, Levchenko et al. 2005;

Goun, Pillow et al. 2006; Mae and Langel 2006; Eguchi, Meade et al. 2009; Heitz,

Morris et al. 2009; Inomata, Ohno et al. 2009). Effective PTD-mediated delivery of peptides, proteins, nucleic acids, antibodies, liposomes, nanoparticles and drugs have all been demonstrated. While many of these cargo types have been delivered in vivo, bioavailability (especially with regard to systemic delivery) has imposed major

113 restrictions on their effectiveness. It is my hope that further elucidation of the molecular mechanism of transduction will support technological advances that allow us to further circumvent Lipinski’s rule of 5 and expedite the clinical use of multiple macromolecular therapeutic modalities (Lipinski, Lombardo et al. 2001).

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