A Thesis Entitled Characterization of Centriolar Protein Poc1 by Anthony R. Cekic Submitted to the Graduate Faculty As Partial F

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A Thesis

Entitled

Characterization of Centriolar Protein Poc1

Anthony R. Cekic

Submitted to the Graduate Faculty as partial fulfillment of the requirements for the

Master of Science Degree in Biology

______Dr. Tomer Avidor-Reiss, Committee Chair

______Dr. Song-Tao Liu, Committee Member

______Dr. Guofa Liu, Committee Member

______Dr. Bruce Bamber, Committee Member

______Dr. Amanda C. Bryant-Friedrich, Dean College of Graduate Studies

The University of Toledo

August 2017

This document is copyrighted material. Under copyright law, no parts of this document

may be reproduced without the expressed permission of the author.

An Abstract of

“Characterization of Centriolar Protein Poc1”

Anthony R. Cekic

Submitted to the Graduate Faculty as partial fulfillment of the requirements for the Master of Science Degree in Biology.

The University of Toledo August 2017

The centrioles are one of the largest protein-based structures in the cell. A typical centriole consists of nine triplet microtubules as commonly depicted in electron microscopy images in textbooks. Centrioles that don’t adhere to the established typical centriole structure are called atypical centrioles. Sperm centrioles in human and Drosophila have been found to contain both typical and atypical centrioles. Human sperms contain an atypical distal centriole and a typical proximal centriole. Drosophila sperms contain a homologous proximal atypical centrioles called the proximal centriole-like (PCL) and an elongated version of the distal centriole called the giant centriole. Centriolar protein Poc1 has been shown to be essential for the stability and function of centrioles in various animal cells.

How Poc1 carries out these functions remains unclear and no adequate tools have been developed to study in this aspect. Here, we show the tools that have been developed to better characterize Poc1 proteins in both Drosophila and human sperms. Firstly, bioinformatics analysis was performed to better understand the Poc1 protein regions in both Drosophila and humans. Secondly, DmPoc1 antibodies were developed and used to observe the localization and protein stability in the sperm centrioles of several DmPoc1 mutants that were discovered through various mutant screens. Using these tools, we found

iii the Poc1 protein regions responsible for DmPoc1 localization and function. Next, several

GFP tagged DmPoc1 protein constructs were generated to better understand the effects of

DmPoc1 regions on its centriole localization and protein stability. Finally, HsPOC1B antibodies were generated to characterize HsPOC1B localization and protein interactions in human sperms. These data suggest that the tools generated are effective and can be used for future research in Poc1 protein characterization.

iv Acknowledgements

There are many people I would like to thank for this work. Foremost is my mentor Dr.

Tomer Avidor-Reiss. You believed in me in a time I didn't even believe in myself. I would also like to thank my friends and colleagues Andrew Ha, Kyoung Jo, Yongheng Wang,

Chris Flora, and Lilli Fishman. You gave me a wonderful lifelong experience. Finally, I would like to thank my parents for supporting me and encouraging me to see this through to the end.

v Table of Contents

Abstract ...... iii

Acknowledgements ...... v

Table of Contents ...... vi

List of Figures ...... Error! Bookmark not defined.

List of Abbreviations ...... xi

1 Introduction ...... Error! Bookmark not defined.

1.1 The Centrioles are Organelles that are Essential in Sperm ...... 1

1.2 Poc1 Protein Family is a Conserved Centriolar Protein Between Drosophila and

Humans ...... 2

1.3 Poc1 Protein Family Localizes Throughout Centrioles and Cilium ...... 5

1.4 Poc1b is Essential for Ciliogenesis, Cilia Length, and Function ...... 7

1.5 HsPOC1A and HsPOC1B are Essential for Proper Embryo and Germline Cell

Development ...... 8

2 Materials and methods ...... 11

vi 2.1 Bioinformatics...... 11

2.2 Transgenic Flies ...... 12

2.3 Plasmid Generation ...... 12

2.4 His-Tag Pulldown ...... 13

2.5 GST-Tag Pulldown ...... 14

2.6 Antibody Generation ...... 17

2.7 Affinity Purification ...... 17

2.8 Tissue Culture and lysates ...... 18

2.9 Tissue Culture Transfection ...... 18

2.10 Antibodies use in Western Blotting and Dot Blotting ...... 22

2.11 Western Blots ...... 22

2.12 Dot Blots ...... 23

2.13 Peptide Blocking ...... 23

2.14 RFP Pulldown ...... 24

2.15 HsPoc1b Immunoprecipitation ...... 25

3 Results ...... 27

3.1 DmPoc1b and HsPOC1A and B Contain Conserved WD and Coiled-coil Regions,

and a Non-conserved Disordered Region ...... 27

3.2 WD domain of Poc1 plays a functional role in spermatozoa and male fertility ...... 32

3.3 DmPoc1 WD+M is Required for Localization to the PCL and Acts Similarly to

DmPoc1a ...... 35

3.4 Antibody 538 is Specific Towards HsPOC1B ...... 37

3.5 Dot blot Analysis Suggests HsPOC1B Antibodies Bind to Highly Charged Areas 38

vii 3.6 Peptides Blocking Determined 537 Epitope is Exclusive to HsPOC1B 382-406 ... 38

3.7 Preliminary Attempts at HsPoc1b IP Show Possible HsPoc1b-HsPoc5 Protein

Interaction ...... 40

4 Discussion and Future Direction ...... 42

4.1 Project summary ...... 42

4.2 Future Directions ...... 43

References ...... 45

viii List of Figures

1. Predicted Poc1 protein regions and structures are conserved across species...... 2

2. Poc1 localizes to specific centriolar substructures, the triple microtubules and the centriole lumen...... 5

3. Antibody Generation Guide ...... 14

4. DmPoc1 and HsPoc1a and HsPoc1b share sites of structural integrity and binding in WD region ...... 24

5. DmPoc1 and HsPoc1 share similar disordered region positions ...... 26

6. DmPoc1b and HsPoc1 proteins have a conserved positioning of coiled-coil regions ...27

7. Bioinformatics data suggests global positioning of WD regions, disordered regions, and coiled-coil regions ...... 28

8. Poc1 WD regions are responsible for localization to the giant centriole and male fertility function ...... 29

9. DmPoc1b WD and disordered regions are required for localization to the PCL ...... 32

10. Anti-HsPoc1b 538 is HsPoc1B-specific ...... 34 ix 11. Monoclonal antibodies show affinity towards peptides B or E, while Polyclonal antibodies show affinity towards E, F, or A ...... 35

11. Preliminary experiments show potential HsPoc5-HsPoc1b interaction ...... 37

x List of Abbreviations

Co-IP ...... Co-immunoprecpitation Cr...... Chlamydomonas reinhardtii

Dm...... Drosophila melanogaster

Hs ...... Homo sapien (human)

IP ...... Immunoprecipitation

Mm ...... Mus musculus (mouse)

Poc1...... Proteome of the Centriole 1 Poc5...... Proteome of the Centriole 5 PONDR ...... Predictor of Natural Disordered Regions PCL ...... Proximal Centriole-Like

Tt ...... Tetrahymena thermophila

WB ...... Western blot/Western blotting WDSP ...... WD40-repeat protein Structure Predictor

The following formatting guidelines are applied in the thesis:

All names begin with the species initials in order to establish which organisms the text is referring to (i.e. Dmpoc1 represents the gene Drosophila melanogaster poc1). Non- mammalian gene names are italicized begin with lower-case letters (i.e. Dmpoc1a). Non- mammalian protein names begin with upper-case letters (i.e. DmPoc1b). Rodent genes are italicized and begin with an upper-case letter (i.e. MmPoc1). Rodent protein names are all xi capitalized (i.e. MmPOC1A). Human genes are all upper case and italicized (i.e. HsPOC1A

) and protein names are just upper-case ( HsPOC1A).

xii Chapter 1

1 Introduction

1.1 The Centrioles are Organelles that are Essential in Sperm

The centrioles are one of the largest protein-based structures in animal cells (Winey &

O'Toole, 2014). The centrioles perform two functions. First, they act as microtubule organizing centers during mitosis. Second, during G0 phase, they migrate towards the cell membrane and form cilia. Throughout the cell cycle, cells have two centrioles, a mother centriole and a daughter centriole. The centrioles typically form a 9-fold asymmetrical cylindrical wall made of microtubules (Azimzadeh & Marshall, 2010; Winey & O'Toole,

2014) (Figure 1). Early centrioles contain two structures, the proximal centriole lumen and the cartwheel. The function of the centriole lumen is unknown. The cartwheel acts as a platform for the formation of the new centriole (Figure 1A and 1B) (Azimzadeh &

Marshall, 2010; Firat-Karalar & Stearns, 2014; Hirono, 2014; Winey & O'Toole, 2014).

As the centriole matures into a mother centriole, the cartwheel disappears and the centriolar lumen moves toward the distal end (Figure 1C and 1D). The function

1 of the centriolar lumen is unknown (Winey & O'Toole, 2014).There are centrioles that deviate from the typical structure which are called atypical centrioles. Sperm cells typically have both typical and atypical centrioles (Atul Khire, 2016; Avidor-Reiss, Khire, Fishman,

& Jo, 2015; Blachon et al., 2009; Blachon, Khire, & Avidor-Reiss, 2014) (Lilli Fishman unpublished). In Drosophila, sperms have elongated versions of typical mother centrioles called the giant centrioles and an atypical version of the proximal centrioles called proximal centriole-like (PCL) (Avidor-Reiss et al., 2015; Blachon et al., 2009). In humans, sperms have a degenerated distal centriole that is homologous to the mother centriole and proximal centriole that is homologous to the daughter centriole (Lilli Fishman unpublished). Poc1B protein has been shown to localize to the centrioles of both Drosophila and human sperm.

1.2 Poc1 Protein Family is a Conserved Centriolar Protein Between Drosophila and Humans

Poc1 is a conserved ancestral centriolar protein that maintains centriole integrity and has been recently implicated in male fertility. Poc1 was discovered by two independent comparative genomics studies, followed by a proteomic study that identified ciliary and centrosomal genes, a protein interaction study that confirmed the existence of Poc1 proteins, and two phylogenetic studies classifying Poc1 as an ancestral centriolar protein

(Avidor-Reiss et al., 2004; Carvalho-Santos, Azimzadeh, Pereira-Leal, & Bettencourt-

Dias, 2011; Hames et al., 2008; Hodges, Scheumann, Wickstead, Langdale, & Gull, 2010;

Keller, Romijn, Zamora, Yates, & Marshall, 2005; Li et al., 2004).

In this study, Drosophila and human cells were used as model organisms. Drosophila (Dm) is a classical model organism used in genetic studies and therefore an ideal candidate to

2 observe the effects of poc1 gene mutations (Jana, Bettencourt-Dias, Durand, & Megraw,

2016). The Dmpoc1 gene encodes two protein isoforms, Poc1a and Poc1b. In humans (Hs)

POC1 is composed of two separate genes; these genes are HsPOC1A and HsPOC1B.

HsPOC1A gene forms three protein isoforms while HsPOC1B forms two sets of protein isoforms; HsPOC1A isoforms 1-3 and HsPoc1b isoforms a-b respectively.

Figure 1 Poc1 localizes to specific centriolar substructures, the triple microtubules and the centriole lumen. Early centrioles contain two structures, the proximal centriole lumen (red) and the cartwheel (black). The function of the centriole lumen is unknown. The cartwheel acts as a platform for the formation of new centrioles (A and B). When a mother centriole matures, the cartwheel disappears and the centriole lumen expands and moves to the distal end. Poc1 has been shown to be essential for the stability of this substructure (C and D).

Generally, Poc1 proteins are composed of three distinct regions: the WD region, the disordered-spacer region, and the coiled-coil region (Figure 2A). The WD and coiled-coil regions are conserved between species, but the disordered-spacer region varies in sequence and length (Fourrage, Chevalier, & Houliston, 2010; Hames et al., 2008; Keller et al., 2009;

Keller et al., 2005). WDSP protein software predicts the Poc1 protein family N-terminus to form a conserved beta-propeller structure (Figure 2B). 3 WD40-repeat proteins are one of the largest protein families making up 1% of human genes in the genome (Wang et al., 2015). WD domains are known to typically form beta- propellers (Smith, Gaitatzes, Saxena, & Neer, 1999). The beta-propeller is a disc-like structure with each blade made up of WD40 repeats. In general, each blade is a four- stranded antiparallel beta-sheet (C. K. Chen, Chan, & Wang, 2011). Beta-propeller proteins can contain four to ten blades. Poc1 is made up of seven blades (Blachon et al., 2014;

Hames et al., 2008; Keller et al., 2009). Proteins with seven blades tend to function as either signaling proteins or structural proteins (C. K. Chen et al., 2011). Since Poc1 is responsible for centriole integrity and centriolar protein localization, this leads to the speculation that

Poc1 may act as both a structural protein for the centriole.

A Poc1 Predicted Protein Regions B Poc1 Predicted Structure WD Region

WD40 WD Linker Disordered Coiled-coil Repeat Region Region Region

Figure 2 Predicted Poc1 protein regions and structures are conserved across species. The following predicted Poc1 protein regions are present across evolution: seven WD domains, a disordered region, and a coiled-coil region (A). WDSP protein prediction software suggests Poc1 proteins form a β-propeller (B).

Coiled-coils consist of two to five amphipathic α-helices that twist around one another to form a supercoil (Lupas, 1996). Coiled-coil regions are known to make up 2-3% of all protein residues (Wolf, Kim, & Berger, 1997). They can perform a wide array of functions that are dependent on the structure of the coiled-coil region in question (Burkhard,

Stetefeld, & Strelkov, 2001). For example, coiled coils in vimentin facilitate filament

4 assembly (Herrmann et al., 2000; Strelkov et al., 2001) or in motor proteins such as kinesin, coiled-coils are used for dimerization as well as processive movement (Kozielski et al.,

1997). We speculate that DmPoc1b and HsPOC1A/B proteins might use their coiled-coil regions to form dimers.

Disordered regions are very common, and account for over half the entire Swiss-Prot database (Dyson & Wright, 2005; Wootton, 1994). These disordered regions are dynamic regions that may form stable structures under specific conditions (Demchenko, 2001). For example, calmodulin is a small protein involved in the regulation of a wide variety of intracellular processes. It was found that calmodulin, when dimerized, uses an unstructured random-coil in solution to attain an a-helical conformation (O'Neil, Wolfe, Erickson-

Viitanen, & DeGrado, 1987). It then loses conformation when it binds to the calmodulin- binding domain of smooth muscle myosin light chain kinase (Ehrhardt, Urbauer, & Wand,

1995). We speculate that the unstructured region of Poc1 may conform to either a WD40 repeat or an additional coiled-coil structures when forming a complex.

1.3 Poc1 Protein Family Localizes Throughout Centrioles and Cilium

Poc1 family proteins tend to localize to specific substructures in the mother centrioles and cilium. In Chlamydomonas (Cr), immunofluorescence and immuno-electron microscopy shows that CrPoc1 proteins localize to both the mother centriole and cilium. In early mother centrioles, Poc1 localizes its entire length including the cartwheel and inner and outer triplet microtubule centriolar walls (Keller et al., 2009) (Figure 1A and 1B).

5 In Tetrahymena (Tt), EM tomographs of Ttpoc1 gene knockout shows that Poc1 is essential for connecting the triplet microtubules in the centrioles as well as stabilizing the centriolar lumen (Meehl, Bayless, Giddings, Pearson, & Winey, 2016; Pearson, Osborn, Giddings,

Beales, & Winey, 2009). Poc1 is also responsible for the mother centriole shape as EM tomographs comparing the twist angles of the centriole in wildtype and poc1 knockout reveal that Poc1 is essential for the helical organization of the triplet microtubules of the mother centriole (Meehl et al., 2016). Furthermore, TtPoc1-GFP reveals that Poc1 proteins are recruited to the new daughter centriole in an age-dependent manner (Galati, Abuin,

Tauber, Pham, & Pearson, 2015). However, it remains unclear how Poc1 performs these functions.

In human Retinal Pigment Epithelial (RPE1) cells, both HsPOC1A-mCherry and

HsPOC1B-mCherry localize to the mother centriole in cilium. In the same cells, immuno-

EM tomographs show that HsPOC1B localizes to various areas of the mother centriole: centriole walls (49%) and cartwheel (28%), as well as the cilium’s transition zone (23%)

(Pearson et al., 2009). These data suggest that Poc1 is an integral part of centriole and ciliary structure.

The Poc1 protein family is essential for centriole stability as well as several centriolar functions. In Tetrahymena, comparisons between Wildtype and Ttpoc1 gene knockout EM tomographs reveal TtPoc1 to be required for the stability of specific triplet-microtubules in mother centrioles (Meehl et al., 2016). In co-depleted HsPOC1A and HsPOC1B cervical cancer cells (HeLa), immunofluorescence showed a loss of centrosomal proteins Sas-6 and

C-NAP1; indicating HsPOC1A and HsPOC1B work together to recruit proteins to the centrioles (Venoux et al., 2013). During mitosis, these co-depletions also resulted in the

6 following centrosomal phenotypes: a loss of centriole integrity, monopolar spindle formation, and unequal spindles (Venoux et al., 2013). In Human RPE-1 cells immunofluorescence of acetylated tubulin in gene knockout of HsPOC1B show that

HsPOC1B protein is essential for cilia formation (Pearson et al., 2009). In summary, these data suggest that Poc1 is essential for the stability of specific centriolar substructures as well as functions such as mitotic spindle formation and cilia formation.

1.4 Poc1b is Essential for Ciliogenesis, Cilia Length, and Function

When cells enter G0 phase, the centrioles migrate to the cell membrane and mother centriole acts as a platform for the formation of cilium; this process is called ciliogenesis.

The Poc1b protein family performs several functions in the cilia across animal evolution.

In Zebrafish (Dr), ciliopathies (diseases resulting from dysfunctional cilia) such as kidney cysts, retinal dystrophy, microcephaly, and cilia shortening were observed in Drpoc1b knockdowns, implicating that DrPoc1b proteins serve a vital role in ciliary retinal function

(Pearson et al., 2009; Zhang, Zhang, Wang, & Liu, 2015). In MmPoc1b shRNA transformed mouse kidney cells (mIMCD3), acetylated tubulin immunofluorescence revealed a significant shortening of cilia; indicating that MmPOC1B is required for cilia length (Zhang et al., 2015). In HsPOC1B depleted RPE1 cells, immunofluorescence of acetylated tubulin resulted in no cilia formation, indicating that HsPOC1B is essential for photoreceptor ciliogenesis (Pearson et al., 2009). In summary, Poc1b proteins serve many roles in cilia including formation, length, and function.

7 1.5 HsPOC1A and HsPOC1B are Essential for Proper Embryo and Germline Cell Development

HsPOC1A plays a role in human embryonic development. For example, human infants born with missense HsPOC1A mutations develop SOFT (Short stature, Onychodysplasia,

Facial Dysmorphism, and hypoTrichosis) syndrome, these symptoms have been determined to be the result of ciliopathies (Barraza-Garcia et al., 2016). There have been four studies identifying Hspoc1a mutations that resulted in SOFT syndrome (Barraza-

Garcia et al., 2016; J. H. Chen et al., 2015; Sarig et al., 2012; Shaheen et al., 2012). A whole genome search for homozygous mutations in nine SOFT patients identifies dysfunctional Hspoc1a in two middle-eastern families (Sarig et al., 2012). All nine had a homozygous missense mutation within the fourth WD domain (L171P) of HsPOC1A. Two other independent studies discovered SOFT patients with additional HsPoc1a mutations;

(p.R81*) which is predicted to truncate the protein after the second WD domain (Shaheen et al., 2012), and p.T120A which is within the third WD domain (Koparir et al., 2015).

Immunofluorescence of acetylated tubulin in fibroblasts of the patient with a HsPOC1A nonsense mutation (p.R81*) showed fewer and shorter cilia, a ciliopathy phenotype

(Shaheen et al., 2012). A fourth study identified a patient with SOFT syndrome and type 2 diabetes. This mutation (p.Q350Rfs*4) is a splice site mutation in the unstructured region of HsPoc1a (J. H. Chen et al., 2015).

HsPOC1B mutations in humans result in classic ciliopathies. In two consanguineous

Middle Eastern families, four affected individuals with the same homozygote missense mutation were identified in poc1b. This mutation (R106P) is in the third WD domain of

HsPOC1B. It is associated with cone-rod dystrophy, involuntary eye-movement

(nystagmus), and reduced vision sharpness (visual acuity) (Roosing et al., 2014) as well as

8 with ciliopathy-related developmental defects such as polycystic kidney disease (Beck et al., 2014). Additionally, a 67 year old Dutch male that is a Hspoc1b trans-heterozygote for a deletion mutation with truncated exons 6 and 7 (c.199_201del/p.Gln 67del) and a splice site mutation with truncated exon 7(c.810+1G>T more info), has milder cone-rod dystrophy, involuntary eye-movement, and reduced vision sharpness that became more severe over time (Roosing et al., 2014). In summary, HsPOC1B defects in Humans result in ciliopathies such as cone-rod dystrophy and polycystic kidney disease indicating that

HsPOC1B function in the human cilia during development.

Defects in sperm centrioles can lead to infertility in animals including humans. This has been suggested to be due to the fact that centrioles are inherited paternally in sperm cells

(Sathananthan, 1991, 1994, 1998; Sathananthan et al., 1996). . Dmpoc1 is essential for mother centriole length and centriolar protein recruitment to the PCL (Atul Khire, 2016;

Blachon et al., 2009). In transgenically altered mice, an insertion of the transposable element Cenpw in the 8th exon of MmPoc1a resulted in a shorter skeleton similar to SOFT syndrome in humans (Geister et al., 2015). In the same study, paraffin sections of

MmPoc1a mutant postnatal testis exhibited progressive germ cell loss, suggesting maturation arrest of testes 14 days post-birth (Geister et al., 2015). Furthermore, spermatogonial stem cell transplants in MmPoc1a mutant mice revealed that the sertoli cells are defective; sertoli cells provide nourishment to sperm cells as they mature (Geister et al., 2015). In summary, these data suggest that Poc1a is essential for embryonic skeletal structure and testes development, but not directly for germline cell development.

Poc1b has been shown to be essential to male fertility. When mutating DmPoc1b, male fertility is reduced (Atul Khire, 2016; Avidor-Reiss et al., 2015). This may be due to a

9 reduction in centriolar protein recruitment to the giant centriole length and PCL in

Drosophila spermatids (Atul Khire, 2016; Blachon et al., 2009). Currently, little is known about the functioning mechanism of Poc1b in reproduction.

10 Chapter 2

2 Materials and methods

2.1 Bioinformatics

Poc1 WD domains were analyzed using the WD40-repeat protein structure predictor

(WDSP) (Wang et al., 2015). Disordered protein regions were analyzed using the Predictor of Natural Disordered Regions (PONDR). Coiled-coil regions were identified using

ExPASy COILS/PCOILS server (Alva, Nam, Soding, & Lupas, 2016; Lupas, Van Dyke,

& Stock, 1991). Protein alignments were performed using MEGA6 software. ClustalW protein alignment was performed under the following parameters: Gonnet matrix, gap penalty 35, gap extension penalty 0.75.

11 2.2 Transgenic Flies Drosophila melanogaster stocks were cultured on standard media at 25°C. In vivo

Drosophila Poc1a-GFP and Poc1b-GFP protein expression, Dmpoc1a and Dmpoc1b cDNA and DmPoc1 protein regions were transgenically inserted at random into the second chromosome using a pUAS vector with UAS promoter and an in-frame C-terminal GFP tag. To ensure the Poc1 proteins were specifically express in the sperm, the bag of marbles

(bam-gal4) gene was co-expressed on the third chromosome. Dmpoc1k245 and

Dmpoc1c06059 were obtained from the Avidor-Reiss collection(Blachon et al., 2009).

Dmpoc1W87X is an EMS mutation obtained from the Zuker collection (Koundakjian,

Cowan, Hardy, & Becker, 2004). Dmpoc1W45R is an EMS mutation from the Avidor-Reiss collection.

2.3 Plasmid Generation

312-406aa HsPOC1B-His plasmid (HsPOC1B 312-406-His) was obtained from the Chad

Pearson collection (Pearson et al., 2009). The 312-406 HsPoc1B gene was inserted into the pQE10 vector, which contains six c-terminal histidines. 312-406aa HsPOC1B-GST plasmid (HsPOC1B 312-406-GST) was generated by inserting the 312-406aa HsPOC1B gene into pET28 vector, which contains a c-terminal GST protein (Lilli Fishman). Next

The HsPOC1A and HsPOC1B genes were inserted into the pIC194 vector, which contains a c-terminal mCherry tag.

12 2.4 His-Tag Pulldown

Buffers

1. Lysis Buffer (1L)

a. 50 mM Tris (6.06 g)

b. 150 mM NaCl (8.76 g)

c. 5% glycerol (50 mL)

d. Complete protease inhibitor cocktail (Roche) (add when needed)

1 tablet/10 mL

2. Wash buffer (1 L)

a. 50 mM Tris (6.06 g)

b. 300 mM NaCl (17.52 g)

c. 5% glycerol (50 mL)

d. 20 mM Imidazole (1.36 g)

pH 8.0

3. Elution buffer (100 mL)

a. 50 mM Tris (0.606 g)

b. 300 mM NaCl (1.752 g)

c. 5% glycerol (5 mL)

d. 150 mM Imidazole (1.02 g)

pH 7.5

Protocol

13 1. Took 6 L Poc1B-His tag E. coli at 25°C in LB broth with 100 μg/mL ampicillin

and grew to optical density between 0.4-0.6

2. Stored at 4°C for 3 h to overnight then added 1mM IPTG and incubated at 20°C for

3 h (50 μL before adding IPTG for future testing)

3. Centrifuged pellet at 12,000 xg for 20 min

4. Discarded liquid and resuspended using lysis buffer

5. Sonciated lysate at 40% for 1min total at 10 sec intervals

6. Centrifuged lysate at 20,000 xg for 30min

7. Equilibrated 100 μL nickel beads with 400 μL lysis buffer three times centrifuging

at 500 xg for 2 min and discard supernatant liquid each time

8. Add lysate to bead slurry and incubate overnight at 4°C save (50 μL for future

testing)

9. Centrifuge at 500XG for 5 min and discard supernatant (50 μL for future testing)

10. Wash beads with 2 mLs of wash buffer three-five times centrifuging at 500 xg each

time and discarding supernatant

11. Add 200 μL elution buffer and incubate at 4°C for 1 h

12. Spin down beads at 500 xg for 1 min and extract supernatant

2.5 GST-Tag Pulldown

Buffers

1. Wash buffer (1 L)

14 a. 50 mM Tris (6.06 g)

b. 300 mM NaCl (17.52 g)

c. 5% glycerol (50 mL)

d. 100 mM LiCl (0.42 g)

e. PH 8.0

2. Lysis Buffer (1 L)

a. 50 mM Tris (6.06 g)

b. 150 mM NaCl (8.76 g)

c. 5% glycerol (50 mL)

d. 0.1% Triton-X (1 mL)

e. PH 8.0

3. Elution Buffer (100 mL)

a. 50 mM Tris (0.606 g)

b. 300 mM NaCl (1.752 g)

c. 5% glycerol (0.50 mL)

d. 40 mM reduced glutathione (1.23 g)

e. PH 9.0

15 Protocol

1. Took 6 L Poc1B-GST tag E. coli at 25°C in LB broth with 100 μg/mL ampicillin

and grew to optical density between 0.4-0.6

2. Stored at 4°C for 3 h to overnight then added 1 mM IPTG and incubated at 20°C

for 3 h (50 μL before adding IPTG for future testing)

3. Centrifuged pellet at 12,000 xg for 20min

4. Discarded liquid and resuspended using lysis buffer

5. Sonciated lysate at 40% for 1min total at 10sec intervals

6. Centrifuged lysate at 20,000 xg for 30min

7. Equilibrated 100 μL nickel beads with 400 μL lysis buffer three times centrifuging

at 500 xg for 2min and discard supernatant liquid each time

8. Add lysate to bead slurry and incubate overnight at 4°C save (50 μL for future

testing)

9. Centrifuge at 500XG for 5 min and discard supernatant (50 μL for future testing)

10. Wash beads with 2 mL of wash buffer three-five times centrifuging at 500 xg each

time and discarding supernatant

11. Add 200 μL elution buffer and incubate at 4°C for 1hr

12. Spin down beads at 500 xg for 1min and extract supernatant

16 2.6 Antibody Generation

Polyclonal antibody Anti-DmPoc1N rabbit was generated by injecting the peptide sequence DmPoc1 6-20 (RDPALERHFTGHSGG) into a rabbit that was then subsequently bled. Peptide generation and injection was performed by Pacific Immunology.

Polyclonal antibody Anti-DmPoc1C rabbit was generated by injecting the peptide sequence DmPoc1a 355-373 (LSEENFQVLDSKHSTQPEK) and DmPoc1b 355-366

(LSEENFQVLD) into a rabbit that was then subsequently bled. Peptide generation and injection was performed by Pacific Immunology.

Polyclonal antibodies Anti-HsPOC1B rabbit 537 and 538 were generated by injecting a

HsPOC1B 312-406-His protein expressed in BL21 E. coli into two separate rabbits. Protein was purified using His-tag affinity chromatography (see His-tag pulldown protocol).

Injection was performed by Pacific Immunology.

Monoclonal antibodies Anti-POC1B rat (M1-M10) were generated by injecting a

Histidine-tagged HsPoc1B 312-406 expressed in BL21 E. coli. Injection and hybridoma formation was performed by Promab.

2.7 Affinity Purification

Polyclonal antibodies 537 and 538 were affinity purified by attaching GST-tagged

HsPoc1b 312-406 protein fragment to a glutathione bead affinity column. Antiseras from the 4th bleed of 537 and 9th bleed of 538 were individually run through the column.

Antibodies that had an affinity to the protein fragment stayed attached while the remaining serum was run through and discarded. Then the antibodies were detached from the protein

17 fragments using a highly charged buffer and collected. Affinity purification was performed by Pacific Immunology.

2.8 Tissue Culture and lysates

HeLa cell cultures were grown in DMEM with 10% FBS and 1% Pen/Strep. Incubation conditions were at 5% CO2 and 37°C. Lysates were made using RIPA buffer: 50 mM Tris, pH 7.5, 150 mM NaCl, 0.1% Triton X-100, 2 mM EGTA, containing freshly added: 1 mM phenylmethylsulfonyl fluoride (PMSF) and EDTA-free protease inhibitor tablets with

1tablet/10mLs (Roche)

Lysate protein concentrations were determined using a bradford assay (Bio-Rad Protein assay dye reagent (cat# 500-0006) and comparing concentrations to a protein concentration curve generated by using BSA solutions with established concentrations (Thermo

Scientific pre-diluted BSA assay standard kit cat# 23208)

2.9 Tissue Culture Transfection

Qiagen effectene transfection (cat# 301425)

1. Transfections were performed in 6-well tissue culture dishes with cells at 80%

confluence

2. Then the following components were added to a 1.5 mL microcentrifuge tube and

incubated at room temp for 5 min: 0.4 μg DNA, 3.2 μL enhancer, 96.4μL EC buffer

18 3. Next, 20 μL effectene reagent was added to the tube and incubated at room

temperature for 10 min

4. Meanwhile, the media from the cell culture is aspirated and washed with 2 mL

PBS/well. Then, the PBS is aspirated and replace with 1.6 mL cell culture media

5. Once complexes are formed in tube, 0.6 mL cell culture media is added, mixed via

pipetting, and added dropwise to well. Dish is then rocked to ensure homogenous

mixing in media and placed in incubator for 48 h.

6. After 48 h, cell culture media is replaced with standard media with 600 μg/mL

geneticin (G418)

7. After a week, cells are checked via fluorescent microscopy for protein expression

Mirus lipofectamine transfection (cat# MIR 5404)

1. Transfections were performed in 6-well tissue culture dishes with cells at 80%

confluence and Lipofectamine reagent was warmed to room temp and gently vortex

before use

2. Then the following components were added to a 1.5 mL microcentrifuge tube and

pipetted gently: 2.5 μg DNA and 250 μL DMEM (No FBS or Pen/strep added)

3. Next, 7.5 μL effectene reagent was added to the tube and incubated at room

temperature for 30 min

4. Meanwhile, the media from the cell culture is aspirated and washed with 2 mL

PBS/well. Then, the PBS is aspirated and replace with 1.6 mL standard media

5. After 30 min, contents from tube are added dropwise to well. Dish is then rocked

to ensure homogenous mixing in media and placed in incubator for 48 h.

19 6. After 48 h, cell culture media is replaced with standard media with 600 μg/mL

geneticin (G418) and is replaced every 3 days

7. After one week, cells are checked via fluorescent microscopy for protein expression

Table 1 Antibody Generation Guide. Clone name represents the name given to these antibodies by the companies these antibodies were generated by. Antibody designation represents the name the antibodies will be referred by in this thesis. Protein Fragments/Peptides corresponds to the protein fragments used to generate the antibodies. Tested applications represent the tested techniques where the antibody successfully identified Poc1 protein. Clone Antibody Protein Tested Name Designation Fragments/Peptides Applications 3217 DmPoc1N DmPoc1 6-20 WB (Pacific Immunology) 3763 DmPoc1a 355-373 DmPoc1C WB (Pacific Immunology) DmPoc1b 355-366 9319 DmPoc1M 88-106 IF (Pacific Immunology) 5G5A7 M1 HsPoc1b 312-406 IF, IP (Promab) 5G5A10 M2 HsPoc1b 312-406 IF (Promab) 5G5D1 M3 HsPoc1b 312-406 IF (Promab) 5D10D3 M4 HsPoc1b 312-406 IF (Promab) 5D10G7 M5 HsPoc1b 312-406 IF (Promab) 5D10G10 (5G10G10) M6 HsPoc1b 312-406 IF (Promab) 6F7C9 (6F719) M7 HsPoc1b 312-406 None (Promab) 6E9D9 (6E9DE) M8 HsPoc1b 312-406 None (Promab) 7C5E6 M9 HsPoc1b 312-406 IF (Promab) 5G5G9 M10 HsPoc1b 312-406 IF (Promab) Polyclonal Anti-HsPoc1b 10537 4AP 537 HsPoc1b 312-406 IF (Pacific Immunology) Polyclonal Anti-HsPoc1b 10538 9AP 538 HsPoc1b 312-406 IF, WB (Pacific Immunology) Polyclonal Anti-HsPoc1b Pearson Pearson HsPoc1b 312-406 IF (Covance)

21 2.10 Antibodies use in Western Blotting and Dot Blotting

Anti-DmPoc1N was used western blotting at a dilution of 1:2,000 and Anti-DmPoc1C was used at a dilution of 1:3,000

Rabbit anti-HsPoc1B 538 affinity purified 9th bleed and 537 affinity purified 4th bleed were used in westerns at a dilution of 1:1,000

Primary antibodies used in dot blots (538, 537, and rat monoclonal antibodies 1-10) were diluted at 1:100 with 5%BSA. Pearson anti-HsPo1B was used at 1:800.

All western blots and dot blots that used secondary antibody donkey Anti-Rabbit HRP

(Santa Cruz) or goat anti-rat (Jackson Immunology), were used at a dilution of 1:10,000.

2.11 Western Blots

8-10% SDS-PAGEs were made and ran in running buffer at 120V for 1.5-2 h. Transfers were performed using nitrocellulose paper at 100 V for 1 h in transfer buffer. Membrane was blocked with 5% BSA for 1 h at room temperature or overnight at 4°C. Primary antibodies were blotted on membrane overnight at 4°C (see antibodies section for dilutions). Membrane was washed after each immunoblot using 0.1% TBST at three times at 15 min intervals. Secondary antibodies were blotted for 1 h at room temperature. Use

Pico chemiluminescent and let sit for 5 min. Liquid was discarded and membrane was exposed to film or used the Azure Biosystems western blot imager in the instrumentation center.

22 2.12 Dot Blots

Native peptides were diluted 1:1 with ultrapure water while denatured peptides had 4X

SDS buffer and were heated at 98°C for 5min. 1 μg of peptide was placed onto a nitrocellulose membrane. Membrane was washed with 0.1% TBST for 2 min or until blue dye ran off denatured peptide dots.

Blocked with 5% BSA for 1 h at room temperature. Placed membranes in nylon bags and add 3-5 mL diluted antibody (see antibodies section for dilution protocols) and incubated at 4°C overnight. Washed three times at 15 min intervals with 0.1% TBST at room temperature. Added diluted secondary antibody and incubated for 1 h at room temperature.

Washed three times at 15 min intervals with 0.1% TBST at room temperature. Placed in nylon bag and added 3-4 mL pico chemiluminescent to membrane and let sit for 5 min.

Emptied liquid and exposed to film or used the Azure Biosystems western blot imager in the instrumentation center for imaging.

2.13 Peptide Blocking

Anti-HsPoc1b 537 was competitively blocked with a concentration of 1:1 dilution of antibody to either Scrambled (S) or peptides: D, E, and F in 5% BSA.

23 2.14 RFP Pulldown

Buffers (HeLa lysate protein pulldown)

1. Lysis buffer/RIPA buffer

a. 50 mM Tris

b. 150 mM NaCl

c. 0.1% Triton X-100

d. 2 mM EGTA

e. pH 7.5

f. added fresh:

i. 1 mM phenylmethylsulfonyl fluoride (PMSF)

ii. EDTA-free protease inhibitor 1 tablet/10mL buffer

Protocol (HeLa lysate protein pulldown)

1. Aspirated media and washed cells three times in 5 mLs 1X PBS and aspirated

2. Placed 1 mL RIPA buffer in plate and scraped cells

3. Pipet lysate into another dish and scraped, repeating process until all dishes

containing the same cell line were scraped

4. Transfer lysate into tube and vortexed tube for 30 sec then placed on ice for 10min

then centrifuge at 16,000 xg for 15 min at 4°C. Transferred supernatant to new tube

and measured protein concentration using Bradford assay: 0.75 mg/mL for initial

trials and 5 mg/mL for final trial

5. Equilibrate 30 μL beads in 500 μL dilution buffer three times and centrifuge at 1000

xg for 2 min discarding the supernatant each time

6. Incubate lysate with 30 μL GFP-Trap agarose beads for 8 hours at 4°C

24 7. Wash beads 3 times with dilution buffer and elute bound proteins by boiling for 5

minutes in 2xSDS-sample buffer

2.15 HsPoc1b Immunoprecipitation

Buffers (HeLa lysate protein pulldown)

Lysis buffer/RIPA buffer

a. 50 mM Tris

b. 150 mM NaCl

c. 0.1% Triton X-100

d. 2mM EGTA

e. pH 7.5

f. added fresh:

i. 1 mM phenylmethylsulfonyl fluoride (PMSF)

ii. EDTA-free protease inhibitor 1 tablet/10 mL buffer

Protocol (HeLa lysate protein pulldown)

1. Aspirated media and washed cells three times in 5mLs 1X PBS and aspirated

2. Placed 1 mL RIPA buffer in plate and scraped cells

3. Pipet lysate into another dish and scraped, repeating process until all dishes

containing the same cell line were scraped

4. Lysate into tube and vortexed tube for 30sec then placed on ice for 10min then

centrifuge at 16,000 xg for 15 min at 4°C. Measure protein concentration using

Bradford: 0.75 mg/mL for initial trials and for final trial 5 mg/mL

25 5. Equilibrate 30μL beads in 500μL dilution buffer three times and centrifuge at 1000

xg for 2min discarding the supernatant each time

6. Add 200 μL dilution buffer to the supernatant, and incubate with 50 μL GFP-Trap

agarose beads for 8 hours at 4°C

7. Wash beads 3 times with dilution buffer and elute bound proteins by boiling for 5

minutes in 2xSDS-sample buffer

26 Chapter 3

3 Results

3.1 DmPoc1b and HsPOC1A and B Contain Conserved WD and Coiled- coil Regions, and a Non-conserved Disordered Region

To better establish the Poc1 protein regions, bioinformatics analysis was performed using software to identify the WD region, the disordered region, and the coiled-coil region. Poc1

WD domains were analyzed using the WD40-repeat protein structure predictor (WDSP)

(Wang et al., 2015). Disordered protein regions were analyzed using the Predictor of

Natural Disordered Regions (PONDR). Coiled-coil regions were identified using ExPASy

Coils server (Alva et al., 2016; Lupas et al., 1991).

WDSP database analysis was used to predict the following: the amino acids that form each blade, the amino acids responsible for maintaining the WD40 repeat structure (H-bonds), the potential binding sites (hotspots), and the native protein structure that is formed. The amino acid sequences of DmPoc1a, DmPoc1b, HsPOC1A, and HsPOC1B were used to determine conservation of WD protein region between Drosophila and human (Figure 3).

According to the WDSP database, DmPoc1a and DmPoc1b share the

27 same WD protein region suggesting similar proteins interact to the WD region in both

DmPoc1a and DmPoc1b (Figure 3A). The WDSP database predicted HsPOC1A and

HsPOC1B to have similar potential binding sites and amino acids responsible for slight differences in amino acid sequence (Figure 3B and 3C). Furthermore, all the Poc1 WD regions are predicted to form a β-propeller structure. Comparisons between Drosophila and human WD regions suggest the blades, H-bonds, and hotspots are conserved. To confirm the H-bond and hotspot amino acids between Drosophila and human were conserved, ClustalW alignment was performed. Alignments indicate that both H-bonds and hotspots are conserved between DmPoc1, HsPOC1A, and HsPOC1B. These data suggest that the amino acids responsible for maintaining WD domain structure as well as the potential binding sites are conserved.

The disordered region in the Poc1 protein family has never been fully established. To identify which amino acids form the disordered region, Both DmPoc1 and HsPOC1 amino acid sequences were analyzed via PONDR. The disordered protein regions between

DmPoc1a (Figure 4A) and DmPoc1b (Figure 4B) are similar, but are at different positions when compared to HsPoc1a (Figure 4C) and HsPoc1b (Figure 4D).

Coiled-coil analysis predicts that protein DmPoc1b contains a coiled-coil region (Figure

5B), but DmPoc1a does not (Figure 5A). Meanwhile, HsPoc1a and HsPoc1b both contain a coiled-coil region (Figures 5C and 5D).

Based on these data a model was made that suggests that despite stark differences in the amino acid sequences, the positions of these regions are conserved (Figure 6). Comparisons between DmPoc1a and DmPoc1b show that both have similar protein regions except

28 DmPoc1b has a longer disordered region and a coiled-coil region (Figure 6A). HsPOC1A isoform 1 and HsPOC1B isoform a have the same protein regions, but the disordered and coiled-coil regions vary in both length and position (Figure 6B). In summary, these data suggest that Poc1 protein regions are conserved.

A DmPoc1a and DmPoc1b WD Domain Prediction Repeats aa Strand d Loop da Strand a Loop ab Strand b Loop bc Strand c Loop cd H bonds Hotspots 1 9-47 ALERHF TGHSGG ITQLRF GPDGA QIATSS TD S TVILWN LNQAA Pentad 2 52-89 RCIRFA SHSAP VNGVAW SPKGN LVASAG HDR TVKIWE PKLR Pentad N64 H80 3 93-131 GVSGEF VAHSKA VRSVDF DSTGH LMTAS DDK SAKIWR VARR Pentad R106 D122 4 135-173 QFVSSF AQQNNW VRSAKF SPNGK LVATAS DDK SVRIYD VDSG NA W146 R148 D164 5 177-215 ECVRTF TEERAA PRQLAW HPWGN MLAVAL GCN RIKIFD VSGS NA R190 6 219-257 QLLQLY VVHSAP VNDVAG HPSGH FLLSGS DDR TIRILD LLEG Triad N232 D248 7 261-299 RPIYTL TGHTDA VNAVAF SRDGD KFATGG SDR QLLVWQ Tetrad N274

B HsPOC1A WD Domain Prediction Repeats aa Strand d Loop da Strand a Loop ab Strand b Loop bc Strand c Loop cd H bonds Hotspots 1 10-48 SLERHF KGHRDA VTCVDF SINTK QLASGS MD S CLMVWH MKPQ Tetrad M39 2 52-90 SRAYF TGHKDA VTCVNF SPSGH LLASGS RDK TVRIWV PNVK Pentad R81 3 94-132 GESTV RAHTAT VRSVHF CSDGQ SFVTAS DDK TVKLWA THRQ Pentad R107 D123 4 136-174 KFLFSL SQHINW VRCAKF SPDGR LVASAS DDK TVKLWD KSSR Pentad W147 R149 D165 5 178-216 ECVHSY CEHGGF VTYVDF HPSGT CIAAAG MCN TVKVWD VRTHR NA F189 M207 6 221-258 LLQHYQ LHSAA VNGLSF HPSGN YLITAS SDR TLKILD LMEG Triad N233 7 262-300 RPIYTL HGHQGP ATTVAF SRTGE YFASGG SDE QVMVWK Tetrad

C HsPOC1B WD Domain Prediction Repeats aa Strand d Loop da Strand a Loop ab Strand b Loop bc Strand c Loop cd H bonds Hotspots 1 8-47 PVLERY FKGHKAA ITSLDL SPNGK QLATAS WD T FLMVWH FKPH Tetrad W38 2 51-89 ARAYRY VGHKDV VTSVQF SPSGN LLASAS RDR TVRLWI PDKR Pentad R80 3 93-131 GKFSEF KAHTAP VRSVDF SADGQ FLATAS EDK SIKVWS MYRQ Pentad R106 D122 4 135-173 RFLYSL YRHTHW VRCAKF SPDGR LIVSCS EDK TIKIWD TTNK Pentad W146 R148 D164 5 177-215 QCVNNF SDSVGF ANFVDF NPSGT CIASAG SDQ TVKVWD VRVN NA F188 N190 6 219-257 LLQHYQ LHSAA VNGLSF HPSGN YLITAS SDG TLKILD LLEG Triad N232 7 261-299 RPIYTL QGHTGP VFTVSF SKGGE LFASGG ADT QVLLWR Tetrad F274 Figure 3 DmPoc1 and HsPOC1A and HsPOC1B share sites of structural integrity and binding in WD region. DmPoc1a and DmPoc1b share the same WD protein region, hence the name DmPoc1 (A). When comparing the WD regions of DmPoc1 (DmPoc1a and DmPoc1b share the same region) to HsPoc1a and HsPoc1b (B and C), there is a similarity between positions of the H-bonds (blue) as well as the predicted binding sites/hotspots (red).

29 A DmPoc1a Disordered Prediction B DmPoc1b Disordered Prediction 100% 100%

l 80% o

l o 80%

r r 50% 50%

20% r r 20%

D D 0% 0% 0 100 200 300 0 100 200 300 400 Residue Number Residue Number C HsPOC1A Disordered Prediction D HsPOC1B Disordered Prediction

r 100% r 100%

i i 80%

80% l

r r 50% 50%

r r 20% 20% r

D 0%

D 0% 0 100 200 300 400 0 100 200 300 400 Residue Number Residue Number Figure 4 DmPoc1 and HsPOC1 share similar disordered region positions. Peaks >0.65 are of the protein. Disordered analysis suggests that proteins DmPoc1a (Figure 4A) and DmPoc1b (Figure 4B) share a globally similar disordered protein region. . DmPoc1a (A) and DmPoc1b (B) share similar disordered peaks between amino acids 317-348 (>0.8). HsPoc1a (C) and HsPoc1b (D) share slightly different positions with their predicted peaks being 311-362 (>0.8) and 329-434 (>0.65) respectively.

30 A DmPoc1a Coiled-Coil Prediction B DmPoc1b Coiled-Coil Prediction

y y

t 100% t 100%

i i

l l

i i

b 80% b 80%

a a

b b 60% o 60% o

r r

P P

l l 40%

i 40% i

o o

C 20% C 20%

- -

d d

e 0% e 0%

l l

i i

o 0 50 100 150 200 250 300 350 400 o 0 50 100 150 200 250 300 350 400 450 C Residue Position C Residue Position Window 14 Window 21 Window 28 Window 14 Window 21 Window 28

C HsPOC1A Coiled-Coil Prediction D HsPOC1B Coiled-Coil Prediction 100% y y 100%

t t

i i

l l i 80% i

b b 80%

a a

b 60% b

o o 60%

r r

P P

40% l l 40%

i i

o 20% o

C C 20%

- -

d 0% d

e e

l l 0%

i i o 0 50 100 150 200 250 300 350 400 450 o 0 50 100 150 200 250 300 350 400 450 C Residue Position C Residue Position Window 14 Window 21 Window 28 Window 14 Window 21 Window 28 Figure 5 DmPoc1b and HsPOC1 proteins have a conserved positioning of coiled-coil regions. Scanning windows are a set of criterion used to measure whether the regions are found locally or globally, meaning these coiled-coils exclusive to the protein or are found in the same proteins of other species. Window 14 (green) predicts the coiled-coils found are exclusive to the sequence being analyzed, window 21 (blue) predicts the coiled-coils found are in a small group of organisms (insects, mammals, etc.), and window 28 (red) predicts the coiled-coils that are found in a majority of organisms. DmPoc1a is predicted to not contain a coiled-coil protein region (A), whereas DmPoc1b is predicted to contain a slightly global coiled-coil region between amino acids around 377-403 that follows other regions, but has significant differences when compared to other organisms (B). However, both HsPOC1A (C) and HsPOC1B (D) contain global coiled-coil regions in amino acids 363-397 and 440-470 respectively.

31 A DmPoc1 Protein Prediction DmPoc1a Disordered 391 1 47 89 131 173 215 257 299 317 348 9 52 93 135 177 219 261 DmPoc1b Disordered Coiled-Coil 403 1 47 89 131 173 215 257 299 317 366 377 9 52 93 135 177 219 261 B HsPOC1 Protein Prediction HsPOC1A isoform 1 Disordered Coiled-Coil 1 56 98 140 182 224 266 308 311 362 363 397 403 17 59 101 143 185 227 269 HsPOC1B isoform a Disordered Coiled-Coil 1 55 99 139 181 223 265 307 329 434 440 470 478 17 58 101 142 183 226 268 Figure 6 Bioinformatics data suggests global positioning of WD regions, disordered regions, and coiled-coil regions. Poc1 WD protein region structure (black) is conserved across evolution. Coiled-coil region structures (green) relatively less conserved than the WD regions. However, the disordered protein regions (orange) show little to no conservation.

3.2 WD domain of Poc1 plays a functional role in spermatozoa and male fertility

To better understand DmPoc1’s role in fertility, a mutant screening was performed generating several mutants (Atul Khire, 2016; Blachon et al., 2009) (Figure 7A). In collaboration with Atul Khire, who observed the effects on DmPoc1 localization patterns in the spermatazoa (Figure 7B) and male fertility, in the background of the following Poc1 homozygous mutants (Figure 7C). W1118 (Con) flies were used as positive controls that have Wildtype Dmpoc1. DmPoc1W45R is a missense mutation that resulted in a 50% reduction in Drosophila male fertility as well as reduced localization to the Centriole.

32 DmPoc1k245 is a missense mutation on the first intron and results in failure of intron to be spliced out, resulting in a truncated version of Poc1 with the seven WD domains and nine additional amino acids which resulted in a 25% reduction in male fertility, but still localized to both the giant centriole and PCL (Atul Khire, 2016; Blachon et al., 2014). DmPoc1C06059 is a p-element in the poc1 gene promoter resulting in a 20% reduction in male fertility, and didn’t localize to either the giant centriole or PCL (Atul Khire, 2016). Finally, DmPoc1W87X is a nonsense mutation that truncates after the 2nd WD domain, which results in complete loss of male fertility and no localization to the giant centriole and PCL (Atul Khire, 2016).

These data suggest that the WD domain of DmPoc1 plays a functional role in spermatozoa as well as a functional role in male fertility.

Since DmPoc1 mutations resulted in reduced male fertility and centriolar localization, the next logical step was to see if protein stability was affected (Figure 7D). Western blotting using DmPoc1-N and DmPoc1-C antibodies showed the following. W1118 DmPoc1 expresses two stable proteins at 42kDa and 44kDa, representing our wildtype protein interactions. DmPoc1W45R expresses two stable proteins as well, suggesting that global protein shape isn’t affected. DmPoc1K245 has a lower expression of endogenous DmPoc1, but also has a truncated mutant at 30kDa when using DmPoc1-N antibody however the same band cannot be seen when using DmPoc1-C confirming this is the truncated product.

DmPoc1C06059 had lower expression of DmPoc1. DmPoc1W87X shows complete elimination of protein expression in DmPoc1. Furthermore no truncated protein product could be found suggesting that the protein product is unstable.

33 Poc1C A DmPoc1 Mutants and Antibodies (Poc1a 355-373) (Poc1b 355-366) c06059 n

r Poc1N (6-20) Poc1M (88-106) t

I Exon 2 Dmpoc1a

Exon 1 K245 poc1 Promoter Exon 2’ Ex.3 Dmpoc1b Disordered WD1 WD2 WD3 WD4 WD5 WD6 WD7 DmPoc1a (43kDa) W45R W87X Disordered Coiled-Coil WD1 WD2 WD3 WD4 WD5 WD6 WD7 DmPoc1b (44kDa)

B Drosophila Spermatazoa Anti-Poc1M Con k245 c06059 N N N N PCL

GC GC PCL DmPoc1 W45R W87X C Poc1 Mutant Fertility Assay 100

d *** e *** N N h

s 50 ***

% D Poc1 Western blot 0 *** anti-Poc1N Con K245 c06059 W45R W87X Con K245 c06059 W45R W87X kDa 50 Poc1 C-term 37 100 N-term K245 **

25 i

e Tub 50 t 50

n *** ***

I *** anti-Poc1C 50 Poc1 0 37 Con k245 c06059 W45R W87X K245 Tub 50 Poc1 K245 Figure 7 Poc1 WD regions are responsible for localization to the giant centriole and male fertility function. Several DmPoc1 mutations were generated as well as antibodies for use in characterizing Poc1 in immunofluorescence (IF) and western blotting (WB) (A) (Atul Khire, 2016; Blachon et al., 2009; Blachon et al., 2014). IF using anti-Poc1M shows that DmPoc1 protein centriole localization in mutants with unaffected WD region, but not with mutants that affect the same region (B) (Atul Khire, 2016). By mating males with Poc1 mutations with Wildtype females, W45R males have significantly reduced male fertility rates when compared to control (C) (Atul Khire, 2016). To determine protein stability WB using anti-DmPoc1N and DmPoc1C shows all mutants generate a stable protein except for W87X, which is predicted to produce a severely truncated Poc1 protein (D).

34 3.3 DmPoc1 WD+M is Required for Localization to the PCL and Acts Similarly to DmPoc1a

The following constructs, developed by Andrew Ha, are Dmpoc1 cDNA fragments with

C-terminal GFP tags, which were generated in eGFP vector with a Gal4 promoter (Figure

8A). DmPoc1a-GFP and DmPoc1b-GFP contain the cDNA of their respective genes. WD-

GFP is Dmpoc1 cDNA representing amino acids 1-298 that is predicted to translate the

WD region. WD+M-GFP is Dmpoc1 cDNA representing amino acids 1-364 that is predicted to be translating the WD and spacer regions while leaving out the c-terminal region. Poc1aC-GFP is Dmpoc1a cDNA representing amino acids 299-391 that translates the predicted spacer and c-terminal region of Poc1a. Poc1bC-GFP is Dmpoc1b cDNA representing amino acids 299-403 that translates the predicted spacer and c-terminal region of Poc1b.

In collaboration with Andrew Ha, the localization of DmPoc1 fragments to giant centriole and PCL of the spermatozoa are as follows (Figure 8B). DmPoc1a-GFP localizes to the giant centriole. DmPoc1b-GFP localizes to the PCL. WD-GFP localizes to neither the giant centriole nor the PCL. WD+M-GFP localizes to the giant centriole. DmPoc1AC-GFP localizes to neither the giant centriole nor the PCL. DmPoc1BC-GFP localizes to neither the giant centriole nor the PCL.

However, when determining if Poc1 fragments were stably expressed, GFP western blotting found the following. Poc1a-GFP and Poc1b-GFP are stably expressed. WD-GFP is expressed and is a stable protein. WD+M-GFP is expressed and is a stable protein at

67kDa. However, both DmPoc1AC-GFP and DmPoc1BC-GFP are not stable proteins.

This data leads us to the hypothesis that the combination of the WD and unstructured regions of Poc1 is responsible for localization to the giant centriole (Figure 8B).

35 A Poc1-GFP Constructs UAS GFP DmPoc1a-GFP (71kDa)

UAS GFP DmPoc1b-GFP (72kDa)

UAS GFP DmPoc1WD-GFP (60kDa)

UAS GFP DmPoc1WD+M-GFP (67kDa)

UAS GFP DmPoc1AC-GFP (37kDa)

UAS GFP DmPoc1BC-GFP (38kDa)

B Poc1-GFP het localization in spermatazoa N

GC N N GC GC DAPI DAPI PCL DAPI Poc1A-GFP 1mm Poc1B-GFP 1mm WD+M-GFP 1mm

GC GC GC

1mm PCL

Poc1A-GFP 1mm Poc1B-GFP 1mm WD+M-GFP 1mm C Anti-GFP western blot Poc1a Poc1b WD+M WD+M 75 Con -GFP -GFP WD WD+M AC BC AC BC

50 37 50 Actin 37 Figure 8 DmPoc1b WD and disordered regions are required for localization to the Giant Centriole. To further characterize the functions of each region; several DmPoc1- GFP constructs were generated (A) (Andrew Ha). When observing localization patterns of heterozygous Poc1 regions to the spermatozoa centrioles, WD+M-GFP was the only Poc1 fragment to localize to the centriole (B) (Kyoung Jo and Andrew Ha). To check for protein stability of the DmPoc1-GFP constructs, western blotting against GFP showed all constructs except DmPoc1BC-GFP were stably expressed (C).

36 3.4 Antibody 538 is Specific Towards HsPOC1B

Several tools were developed to observe HsPOC1B. HsPOC1A-mCherry and HsPOC1B- mCherry plasmids were obtained from Dr. Chad Pearson to test four HsPOC1B antibodies that were developed for western blotting (Pearson et al., 2009). The first two HsPOC1B antibodies developed were rabbit polyclonal antibodies. To test their specificity, first HeLa cells were transfected with either HsPOC1A-mCherry or HsPOC1B-mCherry.

Immunoblotting with anti-mCherry shows the both POC1A-mCherry or POC1B-mCherry constructs are stably expressed (Figure 9A). Anti-HsPOC1b immunoblotting using the rabbit polyclonal antibody 538 shows specificity towards HsPOC1B-mCherry but not

HsPOC1A-mCherry, suggesting specificity towards HsPOC1B Polyclonal antibody 537

(Figure 9B).

A IB:Anti-mCherry B IB:Anti-HsPOC1B 538 HsPOC1B- HsPOC1A- HsPOC1B- HsPOC1A- Con mcherry mcherry Con mCherry mcherry HsPoc1b HsPoc1b HsPoc1a *

Ac-a-Tub Ac-a-Tub

Figure 9 Anti-HsPOC1B 538 is HsPOC1B-specific. HeLa cells were transfected with either HsPOC1A-mCherry or HsPOC1B-mCherry plasmids. Expressions of POC1B- mCherry plasmids were confirmed using anti-mCherry western blotting (A). To confirm HsPOC1B specificity, transfected HsPOC1A and HsPOC1B-mCherry HeLa cell lysate was used and immunoblotted using Anti-HsPOC1B 538 (B).

37 3.5 Dot blot Analysis Suggests HsPOC1B Antibodies Bind to Highly Charged Areas

To identify specifically where the antibodies bind to, several peptides were generated

(peptides A-F) that represent regions of the HsPOC1B fragment used initially during

HsPOC1B antibody generation (Figure 10A). Native (-SDS) and denatured (+SDS) peptides were used for dot blot analysis to determine efficacy of antibodies for both IF and western blotting respectively. HeLa cell lysate (H) was used as a positive control while scramblase (S) was used as a negative control. Dot blots using HsPOC1B rat monoclonal antibodies M1-M3 and M10 (Figure 10B), show antibodies have an affinity towards native peptide B. Dot blots using HsPOC1B rat monoclonal antibodies M4-M6 and M9 (Figure

10C), show antibodies have an affinity towards native peptide E. Dot blots using polyclonal antibodies 537 and Pearson show affinity towards peptides C and F while 538 shows affinity towards peptide A (Figure 10D). Antibodies 7 and 8 showed no affinity to any peptides (Figure 10E).

3.6 Peptides Blocking Determined 537 Epitope is Exclusive to HsPOC1B 382-406

Another dot blot was performed using only peptides E and F dot blotting and peptide blocking antibodies with either: S, D, E, or F. This was done to determine more specifically where the epitopes of 537 were. Peptide blocking shows that the epitopes of antibodies 537 may be in the junction between E and F (Figure 10F).

38 A Res #: 312 327 336 342 351 357 366 372 381 396 406 I I I I I I I I I I I Seq: RNLKRLHFDSPPHLLDIYPRTPHPHEEKVETVEINPKLEVIDLQISTPPVMDILSFDSTTTTETSGR TLPDKGEEACGYFLNPSLMSPECLPTTT Pep: (A)RNLKRLHFDSPPHLLDIYPRTPHPH (B)DIYPRTPHPHEEKVETVEINPKLEV (C)TVEINPKLEVIDLQISTPPVMDILS (D)STPPVMDILSFDSTTTTETSGRTLP (E)TTETSGRTLPDKGEEACGYFLNPSL (F)DKGEEACGYFLNPSLMSPECLPTTT B HsPOC1B Rat Mononclonal Abs- peptide B affinity D HsPOC1B Rabbit Polyclonal Abs M1 M2 M3 M10 537 538 Pearson SDS SDS SDS SDS SDS SDS SDS - + - + - + - + - + - + - + H H H H H H H S S S S S S S A A A A A A A B B B B B B B C C C C C C C D D D D D D D E E E E E E E F F F F F F F C HsPOC1B Rat Mononclonal Abs- peptide E affinity E HsPOC1B Rat Mononclonal Abs- no affinity M4 M5 M6 M9 M7 M8 SDS SDS SDS SDS SDS SDS - + - + - + - + - + - + H H H H H H S S S S S S A A A A A A B B B B B B C C C C C C D D D D D D E E E E E E F F F F F F

F Peptide blocking using Polyclonal Ab 537 *** E F * - 100% +S +D 50% +E +F 25% - +S +D +E +F Figure 10 Monoclonal antibodies show affinity towards peptides B or E, while Polyclonal antibodies show affinity towards E, F, or A. HsPoc1b antibodies were generated against HsPoc1b fragment 312-406. To determine the epitopes of each antibody, several peptides were generated to represent a section of the fragment (A). To determine affinity towards both native (-SDS) and denatured (+SDS) peptides, dot blot analysis was performed. Monoclonal antibodies M1-M3 and M10 show affinity towards peptide B (B). Monoclonal antibodies M4-M6 and M9 show affinity towards peptide E (C). Polyclonal antibodies 537, 538 , and Pearson show affinity towards peptides A,E, and F (D). Monoclonal antibodies M7 and M8 show no affinity towards any peptides (E). Peptide blocking of 537 using peptides scrambled (S), D, E, and F show the epitope is somewhere in the F region.

39 3.7 Preliminary Attempts at HsPoc1b IP Show Possible HsPoc1b- HsPoc5 Protein Interaction

In collaboration with Lilli Fishman co-localization of HsPoc1b and HsPoc5, another centriolar protein, was observed in human mature sperm centrioles (Figure 11A). To determine if Poc1b and Poc5 interact and to see if interactions were influenced by phosphorylation, mCherry Co-immunoprecipitation (Co-IP) was performed using

HsPoc1b-mCherry and HsPoc5-GFP transfected HeLa cells and immunoblotted with anti-

Poc5 and anti-HsPoc1b 538 in either the absence (PI-) or presence (PI+) of phosphatase inhibitors (Figure 11B). Preliminary data showed small amounts of HsPoc1b-mCherry and

HsPoc5 in both conditions, suggesting the interaction is not influenced by phosphorylation

(Figure 11B). Due to instability of transfections, endogenous HsPoc1b Co-IP was performed using Anti-HsPoc1b M1 for IP and anti-Poc5 and anti-HsPoc1b 538 for immunoblotting (Figure 11C). Preliminary data again suggests possible that HsPoc1b and

HsPoc5 interact.

40 A Centrin HsPOC1B-P merge

N N N

PC DC

Centrin HsPOC5

N N N

PC DC

B PI- PI+ POC1B- POC5- POC1B- Poc5- Std Con mcherry GFP Std mcherry Con GFP IP: Anti-mcherry 75 IB: Anti-HsPOC1B 538 PI- PI+ POC1B- POC5- POC1B- POC5- Std Con mCherry GFP Std mCherry Con GFP 75 IP: Anti-mcherry IB: Anti-HsPOC5

C IgG Rat M1 M1 IgG Rat M1 IP IP Ab TL TL IB: Anti-HsPOC1B 50 538 IgG Rat M1 M1 IgG Rat M1 IP IP Ab TL TL 75 IB: Anti-HsPOC5

Figure 11 Preliminary experiments show potential HsPoc5-HsPoc1b interaction. Anti-centrin, Anti-HsPOC1B 537, and anti-HsPOC5 were used to determine localization in human sperm (A) (Lilli Fishman and Rachel Royfman). MCherry Co-IP was performed using HeLa cell transfected with HsPOC1B-mCherry and HsPOC5-GFP. Pulldowns were done in either the presence (PI+) or absence (PI-) of phosphatase inhibitor. Western blotting against centriolar proteins HsPoc1-mCherry and HsPoc5 show potential interaction regardless of phosphatase inhibitor (B). Next, endogenous HsPoc1b IP was performed to confirm HsPOC1B-HsPOC5 interaction (C).

41 Chapter 4

Discussion and Future Direction

4.1 Project summary

The initial objective of this research project was to characterize centriolar protein Poc1 in both Drosophila and humans. The first step was performing bioinformatics analysis by establishing the distinct protein regions in both Drosophila and humans. This involved analyzing the WD regions, the disordered regions, and the coiled-coil regions. Once the

Poc1 protein regions were analyzed, several tools were first generated in Drosophila to allow researchers to investigate the properties of Poc1.

The first tools developed, were polyclonal antibodies used to identify Poc1 in Drosophila.

Western blotting against Poc1 mutant Drosophila testes confirms antibody specificity. The second tools were Drosophila expressing specific protein regions of Poc1 with a GFP tag on the c-terminus. Western blotting with GFP antibody shows that these proteins are stable and being expressed in Drosophila testes.

42 Research then focused on HsPOC1B and thus tools were needed to perform said research.

The first tools generated were HsPOC1B polyclonal antibodies from rabbits. These were to be used for immunofluorescence and western blot studies. Second, HsPOC1A and

HsPOC1B-mCherry tagged genetic constructs were transfected into HeLa cells to determine antibody specificity. Western blot analysis shows that the rabbit polyclonal antibody is HsPOC1B specific. Dot blot analysis was performed and the epitopes of each antibody were identified. Since it was now feasible to generate HsPOC1B antibody, a monoclonal HsPOC1B rat antibody was generated to allow for a near unlimited supply of antibody. Dot blot experiments identified the epitopes of monoclonal HsPOC1B rat antibodies, and immunofluorescence results confirmed the antibodies are HsPOC1B specific. Finally, these tools were implemented to identify protein interactions with Poc1B in human cells. Two preliminary experiments suggest HsPOC1B and HsPOC5 proteins interact. The first attempt involved performing RFP-pulldown using Poc1B-mCherry transfected HeLa cells and western blotting for HsPOC5. The second attempt used the monoclonal HsPoc1B antibody and performed HsPOC1B Co-IP and western blotted for

HsPOC5.

4.2 Future Directions

With the DmPoc1 antibodies and GFP-tagged DmPoc1a and DmPoc1b genetic constructs, performing GFP Co-IP in S2 cells could identify protein interactions. Once DmPoc1- protein interactions are identified, interactions can be confirmed in vivo using the flies with the same construct. Once interactions have been established, DmPoc1 protein fragments

43 can be used to determine where in the cell DmPoc1 protein interactions are taking place.

To see if these interactions are conserved in humans, immunoprecipitation in HeLa and sperm cells using the monoclonal HsPOC1B rat antibodies can be performed.

Alternatively, if one can get an efficient transfection using large quantities of cells, performing RFP Co-IP using HsPOC1A and HsPOC1B-mCherry could be used to achieve the same aim. Once interactions are confirmed immunofluorescence could be used to determine where in the cell these interactions are taking place.

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