A SINGLE MOLECULE STUDY OF CALCIUM EFFECT ON

Ashapurna Sarma

A Thesis

Submitted to the Graduate College of Bowling Green State University in partial fulfillment of the requirements for the degree of

MASTER OF SCIENCE

December 2010

Committee:

Dr. Weidong Yang, Advisor

Dr. Carol Heckman

Dr. Paul Morris

ii

ABSTRACT

Weidong Yang, Advisor

Nuclear pore complexes (NPCs) embedded in the nuclear envelope (NE) is the sole pathway for direct communication between the and the nucleoplasm of eukaryotic cells. NPC allows unregulated passive diffusion of small molecules (< 20 kDa – 40kDa) and facilitated translocation of larger molecules (up to 500 MDa). More and more evidence suggests that calcium stored in the lumen of nuclear envelope and endoplasmic reticulum may further regulate nucleocytoplasmic transport. However with challenges in direct measurements of transport kinetics in the NPC, the calcium-regulated mechanism is still poorly understood. Here single-molecule fluorescence microscopy was used to characterize the permeability of passive diffusion and facilitated translocation under various calcium store concentrations. By snapshots of real-time transient movements of small molecules (10 kDa dextran) and large molecules (97 kDa - β1 (Imp β), through the NPCs, novel features under real- time trafficking conditions were observed that escaped detection by ensemble measurements. It was found that: i) transport rates cannot be used to reflect the change of nuclear pore permeability, which was mistakenly used in previous ensemble experiments; ii) transport rates for both passive diffusion and facilitated translocation can be significantly affected by the store calcium concentrations; and iii) nuclear pore permeability for passive diffusion is affected more by the amount of stored calcium than that for facilitated translocation.

iii

I dedicate this work to my parents Dr. Nirmala and Mr. Makhan Sarma. iv

ACKNOWLEDGMENTS

I wish to express my sincere gratitude to my advisor Dr. Weidong Yang for putting in hard efforts to guide me through my entire master’s degree program. I am extremely grateful for his constant support and guidance right from planning my project to completing my thesis. I would like to thank Drs. Carol Heckman, Paul Morris and Michael Geusz for accepting to be on my thesis committee and for their fruitful guidance. I also wish to extend my thanks to all my instructors who have taught me various courses. I appreciate the support and encouragement from all the members of Yang Lab specially Dr. Jiong Ma for his help in the microscopy setup. I am thankful to my husband Vishal and all my family members for their love and inspiration. v

TABLE OF CONTENTS

Page

INTRODUCTION ...... 1

BACKGROUND AND SIGNIFICANCE ...... 2

MATERIALS AND METHODS ...... 20

EXPERIMENTAL RESULTS …………...... 30

DISCUSSION ………...... 54

REFERENCES ...... 61

vi

LIST OF FIGURES/TABLES

Figure Page

1 Nuclear pore complex structure and its components...... 3

2 The Nuclear Import ...... 8

3 The Nuclear Export ...... 9

4 Structural changes of the NPC in presence and absence of luminal calcium as observed in

previous studies ...... 12

5 Controversial data regarding calcium–mediated nuclear transport as observed in previous

studies ………...... 15

6 Exponential growth of single-molecule fluorescence work...... 18

7 Molecular size of purified Importin β1 protein determined by SDS-PAGE ...... 22

8 Calcium concentrations in the calcium stores, cytoplasm and nucleus of permeabilized

cells under various conditions ...... 32

9 Typical transport events of dextran molecules through the NE ...... 34

10 Import and export time of 10 kDa dextran molecules ...... 36

11 Import and export times of 10 kDa dextran molecules in high-calcium and normal-

calcium condition ...... 38

12 Import and export times of 10 kDa dextran molecules in calcium depleted conditions 39

13 Import and export efficiency of 10 kDa dextran molecules ...... 41

14 Import and export entrance frequency of 10 kDa dextran molecules ...... 42

15 Comparison of transport kinetics between the different calcium-depletion conditions 44

16 Typical transport events of Imp β molecules through the NPC ...... 46

17 Import and export time of labeled Imp β molecules ...... 48 vii

18 Import and export times of labeled Imp β molecules in high/ normal calcium and calcium

depleted conditions ...... 49

19 Import and export efficiency of Imp β molecules ...... 51

20 Import and export entrance frequency of Imp β molecules ...... 53

21 Kinetic constants of entrance frequency, transport time and efficiency and the nuclear

pore permeability ...... 55

Table Page

1 Transport time for 10 kDa dextran molecules ...... 36

2 Transport efficiency for 10 kDa dextran molecules ...... 41

3 Entrance frequency for 10 kDa dextran molecules ...... 43

4 Transport time for Imp β molecules...... 48

5 Transport efficiency for Imp β molecules ...... 51

6 Entrance Frequency for Imp β molecules ...... 53

1

I. INTRODUCTION

Eukaryotic cell is compartmentalized into cytoplasm and nucleus by a double membrane nuclear envelope (NE). synthesized in the cytoplasm needs to be imported into the nucleus and genetic materials transcribed in the nucleus must be exported to the cytoplasm. This bidirectional exchange of biomolecules across the NE, termed nucleocytoplasmic transport, is enabled by thousands of nuclear pore complexes (NPCs) embedded in the NE. The NPCs allow two transport modes: (i) passive diffusion of small molecules (< 20-40 kDa) and (ii) transport receptor-facilitated transport of larger molecules (up to 50 MDa) [1-3]. Recently, much evidences has indicated that both transport modes can be regulated by calcium ion stores in the perinuclear spaces of the NE and the cisternal spaces of the endoplasmic reticulum (ER).

However, the fundamental mechanism of calcium regulated nuclear transport is still poorly understood. The basic mechanism is investigated in this work by employing an innovative single-molecule method. The results obtained by the method provide great insights into this field.

This thesis consists of six sections: (I) Introduction, (II) Background and Significance, (III)

Methods and Materials, (IV) Experimental Results, (V) Discussion, and (VI) References.

2

II. BACKGROUND AND SIGNIFICANCE

Transport of macromolecules across the nuclear envelop is a constitutive process, but any change in of the components in NPC or impairment in any transport factors involved in transport machinery may impede the nucleocytoplasmic transport. Dysfunction in the nucleocytoplasmic transport are related to numerous human diseases including leukemia, , primary biliary cirrhosis and viral infections [2, 4]. Also, disturbances in the cellular environment like intracellular ionic shift will hinder the nucleocytoplasmic transport mechanism, which can directly affect gene expression, signal transduction, and cell development as a whole.

The divalent calcium cation is a major signaling molecule, and alterations in its concentrations disturb the transport pathways leading to cardiac and neurodegenerative diseases as well as cytotoxicity and cell death [5-8]. With such critical significance in human health, it becomes essential to meticulously review the NPC and the transport across it along with the effect of calcium on it.

2.1 The structure of the NPC

NPC structure has been extensively studied in the past several decades. From the 1950‟s, when the first electron microscope (EM) studies revealed that NE is perforated by pores [9, 10], to date, a variety of imaging tools and techniques have been used to deduce the architecture of this transport machine. [11-13]. The 3D structure of NPC shows a basic framework of eight- fold symmetry, as shown in Fig. 1. The core is symmetrical about the plane of the membrane, but the peripheral components are distinct. The scaffold of NPC is divided into three ring elements: a central spoke ring, a cytoplasmic ring and nuclear ring [14]. The cytoplasmic side has eight 50 nm fibrils, and another eight filaments from the nuclear ring join to a distal ring, forming a 3 nuclear basket. There is a central gated channel, with a inconsistent appearance [15]. Refinement of EM techniques to about 6 nm resolution offers more details of this central framework [16].

The central framework has a diameter of 50 nm in the mid-plane [17]. The central cavity is filled by an aqueous meshwork of long - (FG) repeat sequences (Fig 1A). The axial length of vertebrate NPC is almost 200 nm, the total mass is about 125 MDa and are composed of multiple copies of proteins from approximately 30 different gene products [14]

Figure 1: Nuclear pore complex structure and its components. (A) Side view and top view of the NPC, with approximate dimensions of length and diameter. (B) The spatial locations of the disease-associated Nups in the NPC. The FG-Nups (green text), GLFG-Nups (blue text), FXFG- Nups (red text), and non-FG Nups (Black text) identified so far, are shown as they appear in a yeast and human NPC. A is from http://sspatel.googlepages.com/nuclearporecomplex and B is adopted from Ref. [18] with permissions from the publisher (License number 2464250595338)

2.2 The composition of NPC – (Nups)

Characterization of the NPC components is an important step towards understanding the transport mechanism. The building blocks of NPCs are different proteins, known as (Nups). Due to an octagonal symmetry of the structure, there are at least eight 4 copies of each nucleoporins per NPC, localized on both sides of the NE [19]. The Nups are classified into three groups based on their sequence motifs. The first group constitutes approximately 11 Nups. These Nups are characterized by the presence of F-G dipeptide repeat motifs (Fig 1B). Approximately 15 Nups compose the second group which has distinct architectural functions and forms the NPC scaffold structure. The third category is the trans- membrane domain, anchoring the NPC to the NE [11, 14, 20-22]. The components of NPC are not rigidly assembled; some components detach from the NPC every second, while few others are stable throughout the whole cell cycle. [23]. The Nups with FG repeats have a dominant presence - present in the core structure as well as extending from tips of cytoplasmic filaments and the nuclear basket. They are natively unfolded, flexible and mobile within the pore. It is estimated that there are about 3500 FG repeats in a NPC [11]. FG repeats are positioned in a manner so that they can interact with the nuclear transport factors, thus they play a major role in nucleocytoplasmic transport [3].

2.3 Nucleocytoplasmic transport

NPCs are porous to small molecules and ions, while large molecules are assisted by transport proteins requiring an input of metabolic energy to facilitate their translocation through the NPC. For, the two modes of transport through the NPC: The passive diffusion of small molecules is straightforward compared to the facilitated translocation where numerous proteins are involved to effectively assist the cargos in and out of the nucleus [11, 24].

The facilitated nucleocytoplasmic transport system needs four basic constituent and several accessory factors. These basic components are (1) a signal on the cargo, (2) a transport receptor (NTR)//importin-exportin that recognizes the signal, (3) the Nups FG repeat 5 regions that form a meshwork/brushwork barrier inside the NPC, and (4) the nuclear RanGTP

[25].

The first critical component for transport are the proteins (referred in transport processes as cargos) destined to the nucleus containing specific signal in their primary sequences that dictate their destiny termed as nuclear localization signals (NLS), whereas, nuclear export signals

(NES) are required for the proteins that are to be exported out of the nucleus in a specific signal- directed event [3]. Specific sequences and sub-structural context of an NLS or NES defines specificity for binding to different transport receptors [26].

The second component essential for facilitated NPC transport is a special family of proteins, called transport receptors. These receptors distinguish between inert molecules and molecules destined to go through the NPC by recognizing the NLS for import and the NES for export [1, 27]. The major family of transport receptors are the (kaps), also termed , exportins, or transportins. There are more than 20 different kaps that serve different transport pathways, and the conformations of these kaps depend on their specific cargo. Each

Kap passes the NPC in at least two different conformations: cargo-loaded and empty, and these are structurally very distinct [25]. One of the important and largest nuclear transport receptors are karyopherins β proteins or the importin β-like proteins (Imp β). These importins associate with their macromolecular cargo in the cytoplasm, either directly or via adaptor proteins, like the kap α isoforms [26]. Imp β are large proteins, around 97 kDa in molecular weight [28]. CRM1 is another member of Imp β family and exports cargos containing leucine rich-NES into cytoplasm

[2]. The second group of receptors comprises fewer constituents, one among them is nuclear transport factor2 (NTF2). It binds to the GDP-bound form of and mediates import of Ran into nucleus [29]. Some cargos may also need more than one receptor and still others may not 6 need a receptor at all- they interacts with the Nups directly to mediate transport [26, 30].

Although Kaps are the transport receptors for most proteins and RNAs, some bulk mRNA export employs non-karyopherin transport receptors [31]. The composition and constituents of amino acids in these transport receptors make them flexible molecules, stretching and compressing their domains for smooth transport [32]. Thus, with the number of different transport receptors, and the additional conformational complexity added by their cargoes, diverse receptor-specific Nups are required for efficient movement across the pore [25].

The third and the most important component are the FG-Nups which plays an important role in regulating nucleocytoplasmic transport, which essentially can only be accessed by (cargo- loaded) karyopherins/NTRs. The FG-Nups have distinctive tandem sequence repeats of Phe-gly

(FG), Gly-Leu-Phe-Gly (GLFG), and Phe-any-Phe-Gly (FXFG), followed by characteristic spacer sequences. They are anchored in specific NPC locations by their non-FG domains. The

FG domains are natively unfolded, which support multiple binding and facilitate association and dissociation of receptors. The Phe side chains of the hydrophobic FG core bind to the receptors.

The interaction is weak, but sufficiently transient to enable efficient cargo-receptor complex. The comparative transport efficiency of receptor-mediated cargo, which binds to Nups, is faster than its similar sized counterparts without FG binding sites. Moreover, FG-Nup deletion from NPC results in uncontrolled flow of molecules, altering the permeability barrier which is most likely maintained by the hydrophobic interactions between FG Nups [30, 33].

The fourth one in the list of important components for nucleocytoplasmic pathways, is

Ran, a member of Ras family of small GTPase [34]. Interaction between transport receptors and the Ran GTPase controls the directionality. The transport is driven by the asymmetrical distribution of nucleotide-bound Ran, catalyzing the loading and unloading of cargos in correct 7 compartments [2]. Nuclear Ran is GTP bound while cytoplasmic Ran is GDP bound [30]. A Ran regulator, RanGEF, is the GDP-GTP exchange factor, localized in the nucleus to promote GDP disassociation and promoting GTP binding to Ran (Fig 2). When the Ran-GTP leaves the nucleus, it encounters two related Ran-GTP binding proteins, RanBP1 and RanBP2, which along with Ran GTPase-activating protein (RanGAP) induces GTP hydrolysis (Fig 3). As RanGAP is localized to the cytoplasm, this compartment becomes enriched with RanGDP. Nuclear RanGTP binds to importins to release import cargo, and helps to assemble export cargo with the exportins.

In contrast, the RanGDP is unable to bind to Imp β. NTF2 carries RanGDP from the cytoplasm to the nucleoplasm [14]. The nucleotide bound state of Ran affects interaction of kaps with Nups by promoting association and/or dissociation depending on whether it is acting on an importin or an exportin [2].

The classical mechanism of proteins import and export is shown in Figs 2 and 3, and most of the proteins follow those pathways. Furthermore, some studies claim that very large cargos, such as preribosomal complexes and possibly the proteasome, most likely require the

NPC to undergo significant conformational changes, for their transport [25]. With the dynamic nature of transport through a very complex NPC structure, it becomes difficult to understand how NPC structure changes for its numerous transport pathways. Thus, multiple NPC translocation models have been proposed over the years [30]. Virtual gating [35], selective phase [24], and reduction of dimensionality [36] are three major models. Apart from these, a few other models have also been documented such as the affinity gradient, the oily spaghetti or the two gate models [37-39]. 8

Figure 2: Nuclear Import. The nuclear protein import includes several steps as shown above. Formation of the import complex assembly- cargo proteins carrying a classical basic NLS, the receptor molecule importin β, and the adaptor importin α, which bridges between the cargo and receptor molecules, forms a complex. Then, the cargo translocates through the NPC with the help of FG Nup interaction, and on reaching the nucleus the import complex disassembles. Lastly, the importins are recycled. Importin β is recycled by its association with RanGTP, whereas importin α is exported to cytoplasm by a transport receptor-CAS [26, 33, 40, 41]. The Figure is adopted from reference number [2], with permissions from the publisher (License number 2464250082585) 9

Figure 3: Nuclear Export. In nuclear protein export, the transport receptor CRM1 seems to be equivalent to importin β [32, 34, 42] and its interaction with RanGTP and the NES of a cargo protein in the nucleus forms a tri-molecular cargo complex. This complex is exported out of the nucleus to the cytoplasm as shown above. RanGAP stimulates Ran to hydrolyze its bound GTP to GDP. The resulting conformational change in Ran causes dissociation of the cargo complex, releasing CRM1 and Ran GDP. NTF2 recycles RanGDP back to nucleus; CRM1 too is transported back to nucleus for another round of export [40, 42, 43]. The Figure is adopted from reference number [2], with permissions from the publisher (License number 2464250082585)

2.4 Calcium-Mediated Nuclear Transport

Calcium is important for regulation of cell functions and has an impact on nearly every aspect of cellular life, it mostly serve as second messenger in signal transduction. Numerous studies in past two decades have proposed that NPCs and the associated transport are regulated by changes in calcium concentration either within the lumen of the NE, or at the 10 cytosolic/nucleoplasmic face [44, 45]. However, there is a lot of incongruity in the observed both in terms of NPC structural changes and nucleocytoplasmic transport.

In eukaryotic cells, ER forms the major calcium storage for the cell retaining calcium in milimolar concentration ranges [46]. The cisternal spaces are continuous with spaces between the two layers of NE, called the perinuclear space [47]. The nucleus is surrounded by calcium storage compartment, which sequesters and releases calcium in response to intracellular second messengers [48]. Studies have shown that the calcium stores are regulated by calcium channels located on the cytoplasmic and nucleoplasmic sides of the NE [49] and (1,4,5)-trisphosphate receptors (InsP3Rs) is one of them. Binding of inositol 1,4,5-trisphosphate (InsP3) to the channels opens the channels and releases calcium from the stores into the cytoplasm or the nucleus. To maintain the high calcium concentrations in the calcium store, ATP-dependent calcium uptake pumps sequester calcium back into the stores [50, 51]. However, if calcium pump mechanisms are disabled, for example, by thapsigargin (Tg), a specific inhibitor of the calcium ATPase pump, the calcium storage can be reduced. Calcium concentration can also be depleted by a specific calcium ion ionophore or by calcium ion chelators like bis-aminophenoxy ethane-tetraacetic acid

(BAPTA) [52, 53]. Previous research show that luminal calcium depletion leads to NPC structure change [54] as well as alterations in NPC transport. The following are a few consensuses and contradictions regarding calcium levels in the cell with regard to the NPC structure and nuclear trafficking. There is a constant debate on this issue[45].

2.4.1 Role of calcium in NPC structure

Initial studies propose the presence of a central „plug‟, that moves or blocks the channel of NPC in response to changes in the calcium ion concentration in either the or the lumen 11 of the NE [55]. Many argue about the credibility of the central plug of being an integral part of

NPC or cargo caught in transit. The AFM studies reveal distinct asymmetry of two surfaces- with calcium depletion. Higher calcium gave well-defined central pore regions devoid of plugs whereas depleted cisternal calcium stores caused a presence of plugs in the central channel with an increase of central pore occupancy rate [54, 56, 57]. Consistent with this study, several others reported calcium dependent changes in the shape of NPCs [15, 58]. There are also some contradictory results as to how the structure changes with calcium depletion from the lumen; whether there is shift towards the cytoplasmic side [57], the nuclear side [15, 59], or there is a graded displacement of the central mass towards both the sides of nuclear envelop [58, 60], as shown in Fig 4. Apart from above studies, Oberleithner et al. and Vann et al. have presented results showing that the assembly of NPCs was blocked on calcium depletion [61].

The structural change in the NPC, with respect to the depth of the central pore and the diameter of the pore is correlated with the nucleocytoplasmic transport [15, 62, 63]. Different research groups speculate on different ways this NPC conformational change occurs upon calcium release and its influence on the transport of cargoes across the nuclear membrane. Many scientists conclude that the closed or plugged state of the NPC could be responsible for the inhibition of nuclear transport. Another possibility that because of differences in the availability of the FG Nups that line the nuclear pore, [47] the accessibility and binding affinity with the receptors can be altered with structural changes, which can in turn influence the nuclear transport mechanism. Fahrenkrog et al. studied two specific nucleoporins, Nup153 and Nup21, and their location in the nuclear pore, in different calcium concentrations and found that there are changes in the spatial distribution of FG repeats and calcium depletion constraints the distribution of FG 12 repeats [64]. There is a coherent relationship between structure and function of NPC that is calcium dependent.

Figure 4: Structural changes of the NPC in presence and absence of luminal calcium as observed in previous studies. (A) Open configuration and (B) closed configuration of NPC with a plug like appearance by calcium depletion. Both A and B are observation from the cytoplasmic side of oocyte NPC by AFM, modified from Ref [57], with permissions from the publisher (License number 2464251238311) (C) Opening and (D) Closing of nuclear baskets in absence of luminal calcium. C and D are observations from the nuclear side of native Xenopus oocyte NPC by AFM in contact mode, modified from Ref [15], with permissions from the publisher (License number 2464251073435)

2.4.2 Role of calcium in NPC transport

There is also disagreement about the effect of store calcium levels and the transport modes. There are different schools of thought, which can be summed up as three disputes.

Firstly, both passive diffusion and facilitated transport are inhibited when calcium levels are depleted from the cell. Secondly, it is only passive diffusion that is affected and facilitated 13 transport is independent of luminal calcium ion stores in the cell. And the third argument is that calcium level variation has no effect on nuclear transport.

Calcium stores regulating passive diffusion were initially shown by Stehno-Bittel et al. who observed that after depletion of nuclear store calcium by inositol 1,4,5-trisphosphate or calcium chelators, fluorescent molecules conjugated to 10 kDa dextran were unable to enter the nucleus [65]. A similar finding was reported by Perez-Terzic et al. [54]. These results are summarized in Fig 5. Another widely cited study is by Greber et al. who demonstrated that altering the calcium concentration in the lumen of the NE regulates both passive diffusion and facilitated transport through NPCs. Depletion of the NE calcium store was found to attenuate the nuclear influx of proteins bearing a NLS and of non-specific fluorescent dextran molecules [66].

A few studies relate the transport inhibition with calcium depletion to NPC structure [56, 65, 67].

A Nup gp210 that functions to anchor NPCs, projects into the lumen of the NE, where it can sense calcium levels and thereby mediate changes in NPC structure, as experiments conducted with antibody specific for gp210 were found to inhibit both passive diffusion and signal mediated transport into the nucleus [68]. Malviya et al. showed a link between Ca2+-ATPase phosphorylation and transport of intermediate size particles. [69]. Also Perez-Terzic et al. studied nuclear import of histone H1 (~21 kDa) and observed blockage in transport when stored calcium was depleted [54], Fig 5. The above data indicate that NPC function is sensitive to the concentration of calcium within the NE, and that altering luminal calcium concentration can regulate passive, facilitated, or receptor-mediated transport of molecules.

In contrast to reports cited above, there are several reports asserting that transport is independent of luminal calcium stores [52, 62, 70]. In confocal light microscopy studies,

Oberleithner et al. found that the filling state of the perinuclear calcium store had no influence on 14 the passive transport of 10 kD dextran [71], as shown in Fig 5. Strubing et al. used a green fluorescent protein (GFP) fusion construct to study the effect of calcium store depletion on facilitated transport in human embryonic kidney cell line and found no effect on GFP import or export [62] as indicated in Fig 5. The same research group even studied intermediate sized molecules of 27 KDa enhanced- green fluorescent protein (EGFP), which showed no decrease in nuclear permeation when perinuclear calcium stores were depleted. [70].

A different finding related to high calcium levels was reported, where a rise in cytosolic calcium levels increased the influx of photo-activated GFP across the NE hepatocytes [72]. Some studies propose that high concentrations of divalent cation like calcium ions constrain the distribution of specific FG Nups [64], and change the internal diameter of central plug [56].

Although result of studies involving alteration of calcium store concentration lead to interesting questions concerning the role of calcium in nuclear pore permeability and nuclear trafficking.

2.4.3 A New Approach is needed

A new approach is necessary to fill the gaps in the earlier studies and to overcome the limitations in understanding the role of calcium in nuclear transport. There may be several reasons for these discrepancies; one is that different groups use different concentrations of cisternal calcium levels in their experiments [58]. Different techniques used, along with different model systems for the studies add to the complexity. There is also a persistent worry about the experimental conditions such as handling of samples and visualization methods.

15

Figure 5: Controversial data regarding calcium–mediated nuclear transport as observed in previous studies. (A-B) Confocal fluorescent images of fluoresceinated-10 kDa Dextrans in cardiomyocytes, where transport of the molecules is unaffected in presence of calcium-A, and affected in absence of calcium (by using calcium depleting agent Ionomycin)-B. Images A and B are modified from ref [54], with permissions of the publisher (License number 464260736802) (C-D) Confocal images of fluoresceinated-10 kDa dextrans in HM1 cells, where transport of molecules is not affected in the presence or absence of calcium. Images C and D are modified from Ref [70], with permissions of the publisher (License number 2464280000270). (E-F) Confocal fluorescent images of fluorescein-labeled histone in cardiomyocytes, where transport of the molecules is unaffected in presence of calcium E, and affected in absence of calcium (by using calcium depleting agent Tg) F. Images E and F are modified from Ref [54], (with permissions of the publisher (License number 2464260736802) (G-H) Confocal images of GFP labeled glucocorticoid receptor in HM1 cells, where transport of molecules is not affected in either presence or absence of calcium. Images G and H are modified from Ref [62], © 1999 Rockefeller University Press. 16

However, the possible conflicts can be solved, if we overcome one biggest challenge – of using live cells. Previous studies were done in dead or isolated cells. From intact cells, isolated nucleus , nuclear 'ghost' preparations (containing intact nuclear membranes but no nucleoplasm),

[50] to a much recent cell preparation which serves close-to-native state by Stolz et.al [63], there are several ways for sample preparation. But till date there is no in-vivo analysis of calcium mediated NPC transport. A living cell will give a clear picture, to what is actually happening in the trafficking process and also help clarifying the contradictions involved in this field.

Techniques used so far in the previous investigations were atomic force microscopy

(AFM) [57], Transmission Electron Microscopy (TEM), field emission scanning electron microscopy (FESEM) and near-field scanning optical microscopy (NSOM) etc [48]. Other sophisticated techniques are also available which promises to give more depth to these studies at the nanoscopic level, with dynamic imaging and near to native conditions. One such advance is the novel single molecule imaging, which will perhaps overcome the limitations of the earlier studies.

2.5 Single molecule imaging in nuclear transport studies

Understanding of cellular biology is sometimes complicated by the use of conventional ensemble methods, as the biological reactions are based on single molecules. For a better understanding of complex biological processes, single molecule imaging is an efficient technique. With the aid of single molecule technique, biologists can now study variety of biological molecules like DNA, RNA, proteins and large macromolecular complexes in situ.

Single molecule technique used to measure kinetic parameters of a molecule either in an individual reaction step or on a multistep biochemical pathway. The methodology depend on 17 two general approaches: those allowing observation the of single molecule under thermodynamic equilibrium or non-equilibrium conditions and those that study molecular behavior under applied force. Fluorescence microscopy and imaging, which falls under the first type, is helpful in localization, in deducing molecule orientation and in measuring distances and interaction between two molecules. The second approach is used in investigation of mechanical responses to biological systems in combination with methods like optical traps/tweezers, magnetic tweezers, and AFM [73, 74].

In the last 20 years, many research labs have started using single molecule technique and their applications in studying biological processes, so that a new field has emerged - single molecule biology [75]. There has been a exponential growth of single molecule fluorescence work [76], as shown in Fig 6. Single molecule studies range from: exploring properties of biological macromolecules [77-79], to understanding transcription [80, 81], and investigating protein translocations [82, 83]. There has been phenomenal work carried out in understanding the movement of molecular motors by Selvin‟s group using single molecule approach [84, 85].

The striking resolving power of single molecule studies attract many research labs to employ this approach to nuclear transport studies [82, 83, 86] .

Single molecule methods provide unique information on spatial properties and kinetic processes that are otherwise lost by averaging over large populations of unsynchronized molecules. This technique was particularly suited for nucleocytoplasmic transport studies. Using optical microscopy and high-sensitivity CCD cameras, we can image single molecules as diffraction-limited spots, which may be approximated by a two-dimensional Gaussian function.

The position of the particle can be determined with high precision by a fitting process [83, 87,

88]. The localization precision depends on the signal/noise ratio and was found to range up to a 18 few nanometers in current studies [84, 89]. Single molecule detection follows the traces of single molecules and gives significant insight to nucleocytoplasmic transport [87, 90, 91].

Figure 6: Exponential growth of single-molecule fluorescence work. The graph shows the number of publications per year, searched in PUBMED with the keyword “single-molecule fluorescence”, up to the year 2008. Major technical advances are marked in red. Figure adopted from Ref [76].

All previous investigations on calcium effects on nuclear pore permeability or nuclear transport were carried out by the ensemble measurements of the concentration ratio of molecules in the cytoplasm and the nucleus. Detailed information related to the NPC, such as transport time, transport efficiency and spatial locations of transiting molecules, is inevitably missed due to an inability to capture movements of individual molecules within the sub-micrometer-sized 19

NPC. The use of wide- and narrow-field epi-fluorescence SM microscopic techniques to track passive and facilitated transport through the NPC by Yang et. al, shed light on novel features about nuclear transport that escaped detection by TEM, AFM and other ensemble methods.

Single molecule methods are used in this study to get data on kinetics and localization of molecules undergoing transport in various calcium conditions. Passive diffusion of small molecules and facilitated transport of Imp β1 protein is studied under various calcium store concentrations to elicit the role of calcium in nucleocytoplasmic transport.

20

III. MATERIALS AND METHODS

Overview

Protein extraction, purification, concentration and labeling of Imp β1 protein, required for facilitated transport, are reported in the first section. Here, I describe conditions of cell culture, flow chamber set-up and cell permeation, followed by the fluorescence imaging of calcium concentrations under distinct conditions. Then, a step-by-step approach to tracking nuclear transport by single molecule technique is discussed, and localizing nuclear pores and tracking single particles are explained. A brief note on the analysis of experimental data is also included.

3.1 Protein purification

Imp β1 protein for facilitated transport studies was expressed and purified from

Escherichia coli cells. Frozen stocks of E.coli cells expressing N-terminal histidine tagged human Imp β proteins were activated in 5 mL of Luria Bertani (LB) broth with 5µL of ampicillin

(50 mg/mL) and 0.1 g glucose (2%) by growing them overnight at 37oC in a shaker at 225 rpm.

For scale-up, the saturated culture was transferred to 500 mL LB media with 500 µL ampicillin and 10 g glucose. It was then shaken for 5-6 hours in a 37⁰C incubator. The cells were pelleted by centrifugation at 12000 rpm for 20 minutes (min). The pellet was resuspended in 500 mL yeast extract-tryptone media with 500 µL ampicillin, 5mL glycerol (2%) and 12.5 mL ethanol

(2.5%). 250 µL of 0.5M Isopropyl β-D-1-thiogalactopyranoside (IPTG) was added to ensure a higher yield of protein. After incubation for another 5-6 hours, cells were harvested in log phase

(OD of 0.7-0.8), by centrifugation at 12000 rpm for 20 min at 4⁰C. The supernatant was then discarded and the cell pellet was subjected to a protein extraction process. 21

Extraction was carried out by adding 20 mL resuspension buffer (5 mM Tris, 200 mM

NaCl, 5 mM MgCl2, and 10 mM imidazole, pH 8.0) to the cell pellet. Protease inhibitor cocktail consisting of 200 µL of 0.2 mg/mL pepstatin A, 200 µL of 0.2 mg/mL leupeptin, 200 µL of 2 mg/ml trypsin inhibitor, and 200 µl of phenylmethanesulphonylfluoride (PMSF) were added to prevent protein degradation. Further, 1 mM of DNAase was used to degrade unwanted single- and double-stranded DNA. Lastly, 28 µl of β-mercapthethanol was added to break disulfide bonds. This cell suspension was stirred for 30 minutes and then subjected to high pressure homogenization in a French Press (Mini french press cell FA - VWR International) three times with 960 psi pressure. Finally, approximately 3 mL of opulent to clear lysate was obtained. The lysate was centrifuged at 15000 rpm for 25 min, and the supernatant, i.e. protein extract was collected.

The protein extract was purified by Ni-NTA (nickel-nitrilotriacetic acid Superflow by

Qiagen) chromatography. Acetone from the Ni-NTA was removed by repeated washing and spinning, before use. Ni-NTA was mixed with the supernatant and stirred for about an hour. 12 fractions were collected by column chromatography (gravity-flow, BioRad Econo-Pac, 14 cm x

1.5 cm x 12 cm, with porous 30 µm polyethylene bed support column). The first fraction was the flow through of the supernatant mixed with Ni-NTA. The second fraction was from 2.5 mL of resuspension buffer, the third was from 5 mL of wash buffer (5 mM Tris, 10 mM imdazole and

100 mM NaCl, pH 8.0), and fractions 4-12 were 300 µL of elution buffer (5 mM Tris, 250 mM imidazole, 100 mM NaCl, pH 8.0). The fractions containing purified protein were identified by

SDS-PAGE as a single band at 97 kDa (Fig 7).

Fractions with single pure bands on SDS-PAGE gel (Fig 7) were selected for further protein purification with MonoQ and Superdex 200 (Amersham Pharmacia). The purified 22 proteins were concentrated by spinning in a Nanosep filter with a pore size of 10K at 14000 rpm for 5 min. Taking 500 µL of the protein fraction at a time, the final volume was decreased to about 50 µL, after washing each filter with elution buffer. The protein solution was carefully stirred every time to avoid molecules sticking to the filter.

Figure 7: Molecular size of purified Importin β1 protein determined by SDS-PAGE. Importin β1 fractions purified by Ni-NTA and identified by SDS-PAGE as a single band at 97 kDa. Fractions 10, 11 and 12 showing single bands were labeled for use in single molecule experiments.

The final protein concentration was determined by Bradford protein assay using BSA

(bovine serum albumin) as a standard. Protein solutions of different concentrations (from the range of 1 µL/assay to 8 µL/ assay) as well as BSA stock (from 0 mg/ml to 1.0 mg/ml) were prepared with elution buffer. 10 µL of this solution was added to 190 µL of 50:1 Reagent A (2%

Na2CO3, 0.95% NaHCO3, 1% bicinchoninic acid, 0.16% sodium tartrate and 0.4% NaOH, pH

11.25) Reagent B (4% CuSO4) mixture. This 200 µL reaction mixture was incubated at 37⁰C for

30 min and equilibrated at room temperature for 10 min. The measurements of protein were made in a spectrophotometer (Genesys 10 UV scanning) at 562 nm wavelength. The standard 23

BSA plot and the protein concentrations were plotted. Using the Beer-Lambert law, the protein concentration was deduced. The concentrated Imp β protein was labeled with Alexa Fluor 647 with labeling ratio of approximately 3.5 dye molecules per Imp β.

3.2 Cell culture

A HeLa cell line stably expressing the GFP conjugate of POM121 was used for single molecule experiments. HeLa cells were previously genetically engineered to have GFP (green fluorescent protein) fused to the Nup POM121, to show the location of the NE. For optimal growth conditions during experimentation, a fresh culture of HeLa cell line was started from a stock a week in advance and split at least 3 times before the experiment. Cells were grown in a

2 0 25 cm culture flask with 5 mL media and incubated at 37 C in a 5% CO2 incubator for 48 hour.

A day before the single molecule experiment, cell cultures were spread on an autoclaved coverslip placed in a sterile Petri dish. The coverslip was suspended in DMEM (Dulbeco‟s modified Eagle‟s medium, Grand Island, NY) supplemented with 4.5 g/L glucose, 862 mg/L

GlutaMAX-I, 15 mg/mL phenol red, 100 U/mL penicillin, 100 µg/mL streptomycin and 10% newborn calf serum. The volume of a culture was 1 mL per cover slip, uniformly distributed all over the surface. These dishes with cover slips were then placed into the CO2 incubator and kept for 12 hours prior to the experiment. In order for a cell culture to be suitable for a single molecule experiment, the cell confluency on the coverslip should not exceed 70%.

3.3 Flow chamber setup

For microscopic imaging, flow chambers were constructed by using the coverslip with

HeLa cell culture and placing a small coverslip on top of two silicone grease lines acting as spacers. Preferably two chambers were constructed on a single coverslip, to allow two different 24 conditions for the experiment. The HeLa-cell coverslip was drip-dried and mounted with silicon grease (Dow Corning stopcock grease) in a home-machined aluminum frame-temper, which secured the coverslip in the microscope sample holder. Top coverslips – small slivers of glass coverslip cut with a diamond knife to be approximately 6 mm2 - were placed on a lab tissue and two lines of silicone grease were finely placed along the edges of the small coverslips with a 10 mL plastic syringe. These small coverslips were then picked up with pointed tweezers and inverted over the cell-coated coverslip, forming a flow chamber. Several lines of silicone grease were applied in between the chambers to seal them, to inhibit cross contamination. DMEM media was added to the flow chamber, to prevent cells from dying out during this preparation.

After the construction, the bottom of the cell-coated slide was washed with a wetted cotton- tipped swab, twice with distilled water, and then twice with 95% ethanol and air dried.

3.4 Cell permeation

Cells were washed twice with 25µL transport buffer (20 mM HEPES, 110 mM KOAc, 5 mM NaOAc, 2 mM MgOAc, 1 mM EGTA, pH 7.3). A small triangular piece of filter paper was used to facilitate the flow of buffer through the chamber. The cells were then permeabilized for two min with 25 µL 40 µg/mL digitonin in transport buffer. The permeabiliziation step was monitored in a real time by bright field microscopy. The permeabilized cells were washed again with transport buffer supplemented with 1.5% polyvinylpyrrolidone (PVP; 360 kDa). 1.5% PVP was included in all above buffer solutions to prevent osmotic swelling of nuclei.

3.5 Alteration of calcium store concentration in the NE and fluorescence

imaging 25

Permeabilized cells were washed several times by the transport buffer supplemented with

1.5% PVP. For normal calcium concentration in the NE, cells were immediately treated with the transport cargo or calcium indicator dye. To increase the calcium concentration in the NE, cells were incubated with high calcium transport buffer (20 mM HEPES, 110 mM KOAc, 5 mM

NaOAc, 2 mM MgOAc, 1 mM EGTA, and 2 mM CaCl2, pH 7.3) for 15 min. To deplete calcium from the perinuclear spaces of the NE, cells were treated with 10 mM BAPTA (1,2-bis(o- aminophenoxy)ethane-N,N,N',N'-tetraacetic acid, Sigma-Aldrich) and/or with 3 µM, 500 µM, 1 mM or 2 mM thapsigargin (Sigma-Aldrich) with 1.5% PVP for 20 min.

Wide-field epifluorescence microscopy (Olympus IX81) with a HBO mercury lamp and a set of GFP filters was used to take pictures of the fluorescent NE. The green fluorescence emission was collected by a 1.4 NA 100× oil-immersion apochromatic objective (UPLSAPO

100XO, Olympus), filtered by a dichroic filter (Di01 R405/488/561/635-25x36, Semrock) and an emission filter (NF01-405/488/561/635-25X5.0, Semrock). The fluorescence from the GFP was photobleached for few minutes and then 0.5 µM Green-1 calcium indicator (Invitrogen,

Carlsbad, CA) was added to monitor the calcium concentrations in the NE after the above treatments. A 488 nm laser (Melles Griot) was used to excite Green-1, and the fluorescence was collected by a CoolSnap CCD camera (Roper Scientific). The exposure time was 300 ms. The

Slidebook software package (Intelligent Imaging Innovations) and Metamorph (Universal

Imaging, Media, PA) software packages were used for data acquisition and processing.

3.6 Localization of NE

The position of the NE was determined by wide-field epifluorescence microscopy with illumination by the mercury arc lamp. The pixel intensities within a row or column, 26 approximately perpendicular to the NE, were fit with Gaussian. The peak positions of the

Gaussian for a particular set of pixel intensities was considered the NE position for that row and column. The peak positions of a series of such Gaussians were then fit with a second-degree polynomial, yielding the path of the NE within the entire image.

3.7 Localization of NPC

The position of the nuclear envelope (NE) was localized by fitting GFP fluorescence of

POM121, then single NPC was selected by choosing a fluorescent NPC on the equator of nucleus such that the tangent of the NE at the location of this NPC is parallel to the y direction of the Cartesian coordinates (x, y) in the CCD camera. Examining the ratio of Gaussian widths in the long and short axes of the chosen GFP-NPC fluorescence spots revealed ratios between 1.74 and 1.82.

3.8 Single particle imaging and tracking of nuclear transport

Passive diffusion was analyzed by Alexa Fluor 647 - labeled 10-kDa dextran (Invitrogen,

1–1.5 labeling ratio) was diluted to be 0.1 nM -1 nM and added to the flow chamber in a volume of 25 L. To study facilitated transport, 0.1 nM -1 nM Alexa Fluor 647-labeled Imp β was used as a cargo molecule (3-4 labeling ratio). A 35 mW 633 nm He-Ne laser (Melles Griot) and a

120mWArKr tunable ion laser (Melles Griot) were used to illuminate the transport trajectories of labelled molecules with 5 mW laser power (500 kW/cm2). Single molecule trajectories were captured with high-speed Cascade 128+ CCD camera (Cascade 128+, Roper Scientific) at a frame rate of up to 2500 Hz.

27

3.9 Data analysis

3.9.1 Localization precision

To determine the localization precision for immobile molecules or fluorescent NPCs, the fluorescent spot was fitted to a 2D symmetrical or an elliptical Gaussian function. The precision was determined by the standard deviation of multiple measurements of the central point.

However, for moving molecules, the fluorescent spot was fitted to 2D elliptical Gaussian functions, but the localization precision (σ) was determined by an algorithm of

where „N‟ is the number of collected photons, „a‟ is the effective pixel

size of the detector, „b‟ is the standard deviation of the background in photons per pixel, and„s‟ is the standard deviation of the point spread function [89].

To justify the precision obtained by the standard deviation of multiple measurements and the algorithm, the localizations of immobile fluorescent molecules (Alexa Fluor 647-labeled Imp

β1 absorbed on the surface of a coverslip) and fluorescent NPCs were measured on 155 immobile labeled-Imp β1 molecules and 24 NPCs. The two methods yielded a difference of 0.4

±0.1 nm for immobile molecules. Based on 24 NPCs, the two methods generated a difference of

0.3 ± 0.1 nm.

The system error of alignment (σsys) between red and green fluorescence channels is 1.9 ±

0.1 nm, as determined by measuring 230 immobile Alexa647-labeled GFP fluorescent molecules on the surface of coverslip. The co-localization precision of single molecule trajectories (σsm)

2 2 2 and NE (σNE) / NPC (σNPC) was determined by using the equation sm   NE / NPC   sys .

Experimental measurements were reported as mean ± standard error of the mean unless otherwise noted. 28

3.9.2 Transport time

Video frames were analyzed using Metamorph software for the fluorescent cargo molecules that are transported through the NPC. The fluorescent image of the NE or NPC was overlaid onto the video (converted to individual frames) of the cargo molecules and transport events were counted manually. A complete transport event included the total number of frames from where the molecule starts in one compartment (either cytoplasm or nucleus) and ends in the other compartment. Cytoplasm to nucleus and cytoplasm to cytoplasm events were counted as import events and nucleus to cytoplasm and nucleus to nuclease as export events, as explained above. All the events were separately counted and averaged to obtain the import and export times. Such average times were used to generate the histograms. The histograms were fitted with different functions to get a trend in transport patterns.

3.9.2 Transport efficiency

Accurate transport efficiency estimates were obtained only when the single-molecule trajectories analyzed represented cargo molecules that interacted with the NPC. One of three such NE/NPC crossing events was observed for every NPC included in the analysis. The first class consisted of trajectories for which the first and last points are at least 100 nm from the

NE/NPC, and a line between these points crossed the NE/ NPC (entry and exit compartments different). This class was considered to consist of molecules transported across the NE/NPC, i.e complete events. The second class consisted of trajectories for which the first and last points were at least 100 nm from the NE/NPC and both were in the same compartment (cytoplasm or nucleus) and at least one point is within 100 nm of an NPC location. This class of trajectories represented cargo complexes that potentially interacted with an NPC for at least one frame but did not transport i.e. abortive events. The third class consisted of trajectories that did not fall into 29 either class (i.e. the exit compartment was unclear). These were not further considered in transport efficiency calculations, except for a few experimental conditions like highly depleted calcium, where they were termed trapped events. The import efficiency was the percentage of complete import events over the sum of complete and abortive import events. The export efficiency was the percentage of complete export events over the sum of complete and abortive export events.

3.9.4 Entrance frequency

All the video frames to determine the number of cargo particles that were interacting with the NE/NPC and were involved in some kind of event. Using the formula Entrance Frequency

= , where „n‟ is the concentration of molecules near the entrance of pore and „Kin‟ is the entry rate of molecules into the barrier, entrance frequency was calculated. The total number of events including complete events, abortive events and trapped events for some conditions, that occurred in a particular video was taken into consideration in calculating the rate (expressed as events per second) at which these events interacted with the NE/NPC.

30

IV. EXPERIMENTAL RESULTS

Overview

To study calcium mediated nuclear transport, I altered the calcium store concentrations and imaged calcium-induced fluorescence (see 4.1 Calcium concentrations in the calcium stores). Calcium effects on passive and facilitated transport are documented starting with the transport kinetics of small molecules in the second section (see 4.2 Passive diffusion under various calcium conditions). Data on transport time, transport efficiency and entrance frequency are included in this section along with comparison of transport kinetics under calcium- depleted conditions. Transport of Imp β protein molecules through NPC under various calcium conditions is described in (see 4.3 Facilitated translocation under various calcium conditions)

4.1 Calcium concentrations in the calcium stores

To test the effect of calcium on molecule transport through the NPC, calcium concentrations in the calcium stores were manipulated. During the experiments, the digitonin- permeabilized HeLa cells were maintained in 1mM EGTA, which acts as a calcium buffer maintaining free calcium concentration near the physiological resting level of 100 nM [29].

Therefore, the cells in EGTA transport buffer were considered as normal calcium condition for the experiments. The intact nucleus, together with a controlled cytoplasmic environment, provides a greatly simplified transport system that allows us to elucidate the complicated nuclear transport step by step. Previous ensemble and single-molecule experiments conducted in this system have provided great insights in nuclear transport [29, 36, 38, 53, 92, 93]. 31

To reduce the calcium concentration in the stores, I incubated permeabilized cells were incubated in a normal transport buffer with 1mM EGTA, 10 mM BAPTA and various concentrations of Tg (0 µM, 3 µM, 0.5 mM, 1mM and 2mM). Tg was used to inhibit the calcium pumps in the NE, leading to continuous release of free calcium from the stores. This calcium goes into the cytoplasm and the nucleus [94]. The accumulated free calcium was consumed by BAPTA, which is a calcium chelator [45].

Recently it was reported that increased cytosolic concentration of calcium can increase the nuclear permeability for passive diffusion of small molecules in vivo [72]. To test this effect in the permeabilized cells, EGTA was removed from the normal transport buffer and 2 mM

CaCl2 was added to increase the concentration of free calcium in the buffer.

The effect of calcium in the above treatments was monitored by Green-1 fluorescence intensities in the lumen of NE and ER, the cytoplasm and the nucleus (Fig. 8). The fluorescence intensity in the NE was maximum under normal-calcium conditions as expected (Fig 8B). Under high-calcium conditions, there is an increase in calcium concentration in almost every compartment, (Fig 8A). Conversely, the concentrations were gradually reduced to a minimum stable level when the permeabilized cells were incubated with millimolar Tg and BAPTA (Figs 8

C-G). The fluorescence intensities from each condition were normalized by the fluorescence intensity of the NE under the normal calcium condition (Fig 8H). The fluorescence intensities of the Green-1 decreased as the calcium stores were depleted and the depletion was stable in the presence of ≥1mM Tg. 32

Figure 8: Calcium concentrations in the calcium stores, cytoplasm and nucleus of permeabilized cells under various conditions. Fluorescence intensity of Green-1 calcium indicator dye was used to determine the calcium concentrations in (A) high-calcium condition (2 mM CaCl2) (B) normal-calcium condition (1mM EDTA) and (C-G) different calcium-depleted conditions (0-2 mM Tg + 10 mM BAPTA). Scale bar: 20 µm. (H) Normalized fluorescence intensities under various conditions. The fluorescence intensities were normalized based on the fluorescence intensity of the NE under the normal calcium condition.

4.2 Passive diffusion under various calcium conditions

Small molecules (20-40 kDa) passively diffuse through the NPC while being transporting between the cytoplasm and the nucleus. To study the effect of store calcium concentration on the transport of small molecules, dextran labeled with Alexa Fluor 647 dye was used under high, normal- calcium and calcium- depletion condition.

4.2.1. Single molecule imaging of 10 kDa fluorescent dextran molecules

Previously, 10 kDa dextran molecules were found to diffuse through the NPC in approximately 2 ms with a spatial resolution of about 20 - 40 nm when detected by a frame rate 33 of 500 Hz or 1000 Hz. [89]. Such a spatiotemporal resolution allows capture of only one to two diffusion steps of fluorescent molecules through the NPC, which did not suffice to elucidate spatial information for passive diffusion. A better spatiotemporal resolution was required to capture finer steps of dextran molecules within the NPC. To meet the needs, two major improvements were employed: (i) a faster detection frame rate of 2500 Hz to capture more fine diffusion steps; and (ii) a higher illumination optical density to excite more photons from single molecules and attain higher spatial resolution. These improvements allowed me to capture four to five steps of transient diffusion of Alexa Fluor 647-labeled dextran molecules across the NPC using a 633-nm laser light at irradiance of 100 kW/cm2 with a spatiotemporal resolution of 12 nm and 400 µs.

Tracking the spatial trajectories of transiting dextran molecules, under various store calcium concentration conditions, distinguished the steps of diffusion through the NE. All the events where a molecule interacted with the NE from originating and destination compartments were considered. As shown in Fig 9 a series of still images illustrates a typical sequence of transport events. Dextran molecules undergoing import have two destinations: start from cytoplasm and end in the nucleus after diffusing through the NE, i.e “complete import events”

(Fig 9A), or move back to the cytoplasm after interacting with the NE. The latter molecules do not complete the usual import path but return to their originating compartment (the cytoplasm), and so were called “abortive import events” (Fig 9B). The same was true of export events, namely “complete export events,(Fig 9D) and “abortive export events” (Fig 9C). All these events were observed under all the conditions that were used during the experimentation. Such image series contain information on the spatial position of the single molecules with regard to the NE, as well as temporal information on the dwell time of the molecules through the NE. 34

The trajectories shown in right hand panel of Fig 9 illustrate the paths of single dextran molecules starting from the originating compartment and finishing at the destination compartment. These trajectories were then superimposed on the NE. The red and green lines represent 100 nm-distances from the NE on the cytoplasmic and nuclear sides respectively. The molecules which fall within 200 nm distance were considered as having interacted with the pore, as the axial length of the NPC is around 200 nm.

Figure 9: Typical transport events of dextran molecules through the NE. (A) A cytoplasm- to-nucleus event. A single dextran molecule (red spot) started from the cytoplasm, interacted with the NE (green pixel line), and ended in the nucleus. The trajectories of the event (blue dots and lines) were determined and superimposed on the NE (black line). The red and the green lines represent 100 nm-distances from the NE on the cytoplasm and the nuclear side. Numbers denote time. C, the cytoplasm and N, the nucleus. Scale bar: 2 µm. (B) A cytoplasm-to-cytoplasm event. (C) A nucleus-to-nucleus event. (D) A nucleus-to-cytoplasm event 35

4.2.2 Transport time

The transport time in this work, can be identified as the import or the export time. They were defined as the average dwell time of dextran molecules within the NPCs that undergo either import or export. The import time is the average of all the complete import events and the abortive import events counted. And the same is relevant for the average export events. Dwell times of each single event for the dextran molecules in the NE were observed and compared among samples held under different calcium conditions. Under normal-calcium conditions, import and export events generate the same import and export time of 1.7 ± 0.1 ms. When the calcium levels are elevated in the stores the transport time gets faster. The import time was 1.1 ±

0.1 ms and export time was 1.2 ± 0.1, (Fig 10). On the other hand, under calcium depleted conditions, there were longer dwell times within the NPC. With 10 mM BAPTA as the only additive, there was a minor change in transport time, but the calcium depleted-condition with

BAPTA and different concentrations of Tg, caused an increase in transport time (Fig 10). The average import time of cells treated with 0.5 mM Tg was 2.3 ms, and the average with 1 mM Tg was 2.8 ms, which is almost twice the time taken under normal calcium condition. The import and export times are listed in Table 1 for all the calcium conditions. 36

Figure 10: Import and export time of 10 kDa dextran molecules. Comparison of the import and export times between high- and normal-calcium and calcium-depleted (with 0-1mM Tg + 10 mM BAPTA) conditions. For high- and normal-calcium and calcium-depleted (0 and 3 µM Tg + 10 mM BAPTA) conditions, the dwell time fitting average is considered. *Average time is presented as these conditions have two import and export times, (see Table 1)

Table 1: Transport time for 10 kDa dextran molecules. The import and export times of dextran molecules under the conditions of high and normal-calcium and calcium-depleted (with 0-1mM Tg and 10 mM BAPTA) is listed. τ represents the transport times fitted with mono exponential data for high and normal-calcium conditions. T is the Gaussian fitted transport times for all the calcium-depleted conditions. For highly depleted calcium conditions (0.5 mM and 1 mM Tg + 10 mM BAPTA), there were shorter (T1) and longer (T2) transport times, following two Gaussian functions. The numbers in parentheses indicate the number of events recorded for that condition. 37

The dwell time histograms gave better insight to the transport times. At normal-calcium, the histogram of dwell time of dextran molecules within the NPC follows a mono-exponential decay, and the import as well as export time τ was 1.7 ± 0.1 ms, (Figs 11 C-D). Even with the high-calcium condition, the import time and export time events followed a mono exponential decay (Figs 11 A-B). Under calcium-depleted conditions, the dwell times within the NPC did not follow a mono-exponential decay but rather had a Gaussian distribution (Fig 12). In cells treated with all different calcium depleting agents at various concentrations, there was a deviation in dwell times and their distribution. Cells treated with 10 mM BAPTA and 3 µM Tg showed a single Gaussian distribution, even though the difference in transport time was very small (Fig 12

A-B). When cells were treated with higher doses of Tg, the transport events showed two dwell times, which followed two Gaussian functions. There was a shorter and longer import and export times are listed in Table 1, and they are shown in Fig 10. For the highly depleted calcium conditions, the histogram with two Gaussian functions is shown (Fig 12 C-D). 38

Figure 11: Import and export times of 10 kDa dextran molecules under high-calcium and normal-calcium condition. (A-B) Import and export events of transiting molecules under high calcium condition fitted by a mono exponential function. (C-D) Import and export time of transiting molecules under the normal calcium condition. τ is the time when the histogram decays to half of the maximum number of the events. Bin size: 0.4 ms 39

Figure 12: Import and export times of 10 kDa dextran molecules under calcium depleted conditions. (A-B) Import and export events of transiting molecules under 3 µM Tg + 10 mM BAPTA treatment fitted by a Gaussian function. T is the fitted peak time. Bin size: 1 ms. (C-D) Import and export events of transiting molecules under 1 mM Tg and 10 mM BAPTA treatment fitted by two Gaussian functions. t1 and t2 are the two fitted peak times. Bin size: 0.4 ms 40

4.2.3 Transport efficiency

The import or export efficiency is defined as the percentage of the complete import or export events over the sum of the complete and abortive events. The molecules start from one compartment and move to the other compartment or come back to the same compartment. The efficiency of transport varied with different calcium conditions. Under the normal-calcium condition import and export efficiencies were about 50% (Fig 13). The results agree well with some previous measurements [82]. Under the high-calcium condition, relative transport efficiencies increased up to around 60%, with more complete events than abortive events.

However, under the calcium-depleted condition, most dextran molecules were aborted, which resulted in significant reduction in import or export efficiencies. For calcium depleted conditions more abortive import (from cytoplasm to cytoplasm) and abortive export (from nucleus to nucleus) events (Fig. 9 B andC) were observed compared to complete import (from cytoplasm to nucleus) or complete export (from nucleus to cytoplasm) events, Fig 6 A and D. The change in transport efficiency was not very evident in cells treated with BAPTA alone, but as the Tg concentrations were increased, depleting calcium, the efficiency significantly decreased and there was a striking decline in transport efficiency. 4% import and 6% export efficiencies for the calcium-depleted condition with 1 mM Tg and 10 mM BAPTA (Fig 13). The type of events recorded showed a difference in the calcium-depleted condition, as the export events were less than the import events. When cells were treated with 0.5 mM or 1 mM Tg + 10 mM BAPTA, the export events were only a third of total events observed. 41

Figure 13: Import and export efficiency of 10 kDa dextran molecules. Comparison of the import and the export efficiencies between high- and normal-calcium and calcium-depleted (with 0-1mM Tg and 10 mM BAPTA) conditions. There was a decrease in efficiency from high to, normal to calcium depleted- conditions. Transport efficiency in1 mM Tg with 10 mM BAPTA is significantly low.

Table 2: Transport efficiency for 10 kDa dextran molecules. The import and export efficiencies of dextran molecules under high- and normal-calcium and calcium-depleted (with 0- 1mM Tg and 10 mM BAPTA) conditions. The numbers in parentheses indicate the number of events recorded for that condition. 42

4.2.4 Entrance frequency

The import or export entrance frequency refers the number of molecules interacting with the NE per second. The complete, abortive and trapped events were taken into consideration for both import and export entrance frequency. Trapped events are those events which start from one compartment, interact with the NE and get trapped in the NPC, so that the destination compartment is not known. The entrance frequency is expressed as events per second. Under the normal-calcium condition, the entrance frequencies for both import and export of dextran molecules were approximately the same namely 25 events per second (Fig 14).

Figure 14: Import and export entrance frequency of 10 kDa dextran molecules. Comparison of the import and export times between the high and normal-calcium and calcium-depleted (with 0–1 mM Tg and 10 mM BAPTA) conditions. There was a decrease in efficiency from high to normal to calcium depleted- conditions. Entrance frequency of 1 mM Tg with 10 mM BAPTA is significantly low.

43

Table 3: Entrance frequency for 10 kDa dextran molecules. The import and export efficiencies of dextran molecules under high-calcium, normal-calcium and calcium-depleted (with 0-1mM Tg and 10 mM BAPTA) conditions. The numbers in parentheses indicate the number of events recorded for that condition.

Under calcium-depleted conditions, 10 kDa dextran molecules, showed a lower entrance frequency. The frequency decreases with reduced concentration of calcium in the stores. (Table

3). Under one of the calcium-depleted conditions (1mM Tg and 10 mM BAPTA), the entrance frequency dropped to 2-3 events per second, and the majority of dextran molecules failed to interact regardless of whether the molecules approached from the cytoplasm to the NE or the nucleus to the NE.

4.2.5 Transport kinetics under the calcium-depleted condition

The effect of calcium-depletion was an important aspect of this study as previous results by different scientific groups showed conflicting results. Three transport kinetics, transport time, transport efficiency and entrance frequency were examined under calcium- depleted conditions. 44

A series of Tg concentrations was used (ranging from 0 - 1 mM + 10 mM BAPTA) to study its effect on transport kinetics. As shown in Figure 15, all three parameters followed a linear gradient change with the Tg concentration, but average transport efficiency (Fig 15B), and average entrance frequency (Fig 15C), were changed more dramatically than average transport time (Fig 15A). Specifically, 1.2 ms increase in transport time, 38% decrease in transport efficiency and 14 fewer interaction events for entrance frequency were caused for every increase of 1 mM Tg. The amount of calcium in the stores plays a major role in determining the transport kinetics.

Figure 15: Comparison of transport kinetic parameters between different calcium- depletion conditions. (A) Dependence of transport time on Tg concentration in the presence of 10 mM BAPTA. The points from 0.5 µM to 1 mM were fitted by linear function to obtain 1.2 ms per 1 mM Tg. (B) Dependence of transport efficiency on Tg concentration in the presence of 10 mM BAPTA. The points from 0.5 µM to 1 mM were fitted by linear function to obtain 38% per 1 mM Tg. (C) Dependence of entrance frequency on Tg concentration in the presence of 10 mM BAPTA. The points from 0.5 µM to 1 mM were fitted by linear function to obtain 14 events per 1 mM Tg.

4.3 Facilitated translocation under various calcium conditions

Large molecules are transported through the NPC with the help of transport receptors.

Importin β1 proteins (Imp β) are one of the major transport receptors involved in facilitated transport. Imp β associate with the signal carrying proteins to form a cargo complex, which docks on components of the NPC and translocates through the pore. The FG-Nups in the NPC 45 provide binding sites to Imp β, and their interactions control the passage of the cargo proteins. To study the effect of store calcium concentration on Imp β regulated facilitated transport, Imp β labeled with Alexa Fluor 647 were used under various calcium concentrations. For facilitated transport single molecule experiments under high-, normal and calcium- depleted conditions

(with 0 µM, 3 µM Tg and 1 mM Tg in addition with 10 mM BAPTA) were conducted.

Single molecule studies require the protein to be very pure. Imp β was purified and identified by SDS-PAGE as a single band at 97 kDa, (Fig 7). The purified proteins were then concentrated and labeled with Alexa Fluor 647 and used at 0.1 nM concentration in single molecule experiments.

4.3.1 Single molecule imaging of labeled Imp β molecules

The method of single point illumination and detection was used for these experiments. A

CCD camera was used to image the diffraction-limited spot and a laser beam was shifted off the center of the objective and focused into an inclined illumination volume in the focal plane to reduce the illumination volume in the axial direction. The microscopic set up described in Ma et.al is designed to image only a single fluorescent NPC and to track fluorescent molecules transiting through this NPC [91], as shown in (Fig. 16). An incident 488-nm laser beam is focused through the center of the objective to excite a single GFP-NPC. The centroid of a well- isolated GFP-NPC can be determined from a 2D elliptical Gaussian function. This improvement permitted a precise localization of a single NPC, rather than the entire NE as done in the passive diffusion experiments. For exciting single transiting molecules through the illuminated single

NPC a second laser (633 nm) was used. Maximum photon counts were generated from a single labeled molecule within a sub millisecond detection time by a very high optical density in the 46 illumination volume. A 400-μs detection time and an average optical density of 500 kW∕cm2 in the illumination area, were used to obtain around 1,100 photons. For labeled Imp β, a localization precision of ≈9 nm was obtained for immobile molecules and a localization precision of ≈10 nm was obtained for moving fluorescent molecules when the S/N ratio is ≈11

[91].

Figure 16: Typical transport events of Imp β molecules through the NPC. (A) A cytoplasm- to-nucleus event. A single Imp β molecule (red spot) started from the cytoplasm, interacted with the NPC (green spot), and ended in the nucleus. Numbers denote time. C, the cytoplasm and N, the nucleus. Scale bar: 1 µm. (B) A cytoplasm-to-cytoplasm event. (C) A nucleus-to-cytoplasm event. (D) A nucleus-to-nucleus event.

The transient transport of the single Imp β molecule through the single NPC in the permeabilized HeLa cells stably was imaged and tracked (Fig 16). Tracking the spatial trajectories of individual transiting Imp β molecules, under various store calcium concentration 47 conditions, distinguishes the finer steps of facilitated transport through a single NPC. Similar to the dextran molecules, the Imp β molecules undergo import events, export events, and abortive events. All the events where molecules interacted with the NPC from originating and destination compartments were considered. The trajectories of Imp β molecules around the area of NPC, at various calcium concentrations, were analyzed to determine transport time, efficiency and entrance frequency.

4.3.2 Transport time

The transport time is the average dwell time of Imp β molecules that undergo either import or export within the NPCs. Similar to passive diffusion of 10 kDa dextran molecules, the average time of each single event for Imp β molecules in the NPC were calculated and compared under different calcium conditions. Under the normal-calcium condition, Imp β1 molecules were transported through the NPC with almost the same import and export times. The average import time was 3.2 ± 0.3 ms and the export time was 3.8 ± 0.3 ms. When the calcium levels are elevated in the store, the average transport time increased more than two-fold to 1.1 ± 0.1 ms.

When calcium stores were depleted with different concentrations of Tg and BAPTA, the average transport times were also faster. The dwell time of molecules within the NPC was shorter, under calcium depleted conditions, whether they were undergoing complete or abortive events. The depleted condition with only 10 mM BAPTA had the shortest average import time of 1.3 ± 0.1; while other calcium depleted conditions (3 µM Tg and 1 mM Tg with BAPTA) caused the times to increase (Fig 17). The average export times under calcium depleted conditions showed the same trends (Table 4). The dwell time histograms obtained for Imp β molecules under various calcium conditions follow mono-exponential decay as shown in Fig 18. 48

Figure 17: Import and export time of labeled Imp β molecules. Comparison of the average import and the export times between high and normal-calcium and calcium-depleted (with 0 - 1mM Tg and 10 mM BAPTA) conditions.

Table 4: Transport time for Imp β molecules. The import and export times of labeled Imp β molecules under high andnormal-calcium and calcium-depleted (with 0-1mM Tg and 10 mM BAPTA) conditions. 49

Figure 18: Import and export times of labeled Imp β molecules under high and normal calcium and calcium depleted conditions. (A-B) Dwell time histograms of import and export events of transiting Imp β molecules under the high calcium condition. (C-D) Dwell time histograms under the normal calcium condition. (E-J) Dwell time histograms under different calcium depleted conditions. All histograms are fitted by a mono exponential function and τ is the time when the histogram decays to half of the maximum number of the events. Bin size: 0.4 ms. 50

4.3.3 Transport efficiency

The import or export efficiencies, as defined in the section on passive diffusion of 10 kDa dextran molecules, varied with different calcium conditions. Under the normal-calcium condition, import and the export efficiencies were determined to be 50 ± 5% import efficiency and 47 ± 4% export efficiency. The results agree well with some previous measurements [88].

Under the high-calcium condition, relative transport efficiencies were 53 ± 3%, almost the same as normal calcium condition. However, under the calcium-depleted condition, the transport efficiency decreased to 10 – 20 %. Most Imp β molecules transit events were aborted, resulting in significant reduction in import and export efficiencies. Thus, the high calcium condition showed little effect on transport efficiencies compared to normal, but due to increases in abortive events, under calcium depleted conditions, the efficiencies declined. Even when cells were treated only with 10 mM BAPTA there was a significant decrease, almost to half of the transport efficiency in normal calcium condition. For 3 µM Tg and BAPTA treated cells the transport efficiency further decreased and in 1 mM Tg and BAPTA the transport efficiency was almost 10

± 2%, (Fig 19). The number of events recorded for export was always less compared to the import events, for some of the calcium depleted conditions as shown in Table 5. For example, when cells were treated with 3 µM and 1 mM Tg + 10 mM BAPTA, total export events are only a half of total import events. 51

Figure 19: Import and export efficiency of Imp β molecules. Comparison of the import and the export efficiencies between high and normal-calcium and calcium-depleted (with 0 - 1mM Tg and 10 mM BAPTA) conditions. There was a decrease in transport efficiency from high and normal to the calcium depleted condition.

Table 5: Transport efficiency for Imp β molecules. The import and export efficiencies of Imp β molecules under high and normal-calcium and calcium-depleted (with 0-1mM Tg and 10 mM BAPTA) conditions. The numbers in parentheses indicate the number of events recorded for that condition. 52

4.3.4 Entrance frequency

The import or export entrance frequency refers the number of molecules interacting with the single NPC per second. Under the normal-calcium condition, the entrance frequency for both import and export of Imp β molecules was 22 events per NPC per second,(Fig 20). Under the high-calcium condition, the import entrance frequency was about 20 events per NPC per second, while the export entrance frequency was 30 events per second. Thus, the rates at which these interactions occur with the NPC are similar for these two calcium conditions. But Imp β molecules under calcium-depleted conditions showed strikingly different entrance frequencies, following unusual trends. Decrease in store calcium with only 10 mM BAPTA did not cause a large difference in the entrance frequency when compared with the normal condition, with only a decrease to about 17 events per NPC per second. But when cells were treated with 3 µM Tg + 10 mM BAPTA, the import entrance efficiency escalated to 60 events per NPC per second. With higher levels of Tg (1mM Tg + 10 mM BAPTA), the cells showed 45 import events per NPC per second. The export entrance frequency, however, did not show such a marked difference (Table

6). The high rate of import entrance frequency when store calcium was depleted suggests a higher probability of Imp β molecules interacting with the NPC from the cytoplasm. The time interval between these events was less, compared to the molecules interacting with the NPC from nucleus. The nature of the Imp β molecules and their binding efficiency with the FG-Nups may be responsible for the remarkable difference of entrance frequency under calcium depleted conditions. 53

Figure 20: Import and export entrance frequency of Imp β molecules. Comparison of the import and the export times between the high- and normal-calcium and the calcium-depleted (with 0 - 1mM Tg and 10 mM BAPTA) conditions.

Table 6: Entrance frequency for Imp β molecules. The import and export efficiencies of Imp β molecules under high and normal-calcium and calcium-depleted (with 0-1mM Tg and 10 mM BAPTA) conditions. The numbers in parentheses indicate the number of events recorded for that condition. 54

V. DISCUSSION

Single-molecule investigations of calcium-regulated passive diffusion and facilitated translocation through the NPCs have provided great insights into the field of nuclear transport.

Single-molecule methods enable us to elucidate the mechanisms proposed based on previous ensemble methods. By single-molecule methods, it was found that: i) transport rates cannot be used to reflect the change of nuclear pore permeability, which was mistakenly assumed in previous ensemble experiments; ii) transport rates for both passive diffusion and facilitated translocation can be significantly affected by the calcium store concentrations; and iii) nuclear pore permeability for passive diffusion is affected more by the amount of stored calcium than that for facilitated translocation.

5.1 Nuclear transport rate, nuclear pore permeability and transport kinetics

Single-molecule measurements of transport time, efficiency and entrance frequency for passive diffusion and facilitated translocation contributed to the existing understanding of the dependence of nuclear transport rate and nuclear pore permeability on the calcium concentrations in the calcium stores. As shown in Figure 21, the steady state flux of mobile molecules through the permeable barrier within the NPC can be expressed as:

d  I DA  DA NC (eq. 1) dx L

( in C out C () out N  in N )ACkkAkCkI (eq. 2)

 CCPI NC (eq. 3) Where: I current of particles crossing the area, A, each second D diffusion constant L length of the nuclear pore

in ,kk out entry and exit rate through the barrier

, NC the concentration of the particles within the pore on the left and right sides 55

,CC NC the concentration of the particles in the cytoplasm and the nucleus P the permeability of NPC

Solving the equation for I by eliminating C and N :

ADkin I   CC NC )( (eq. 4) 2D  Lkout ADk P  in (eq. 5) 2D  Lkout Definitions of entrance frequency, transport time and efficiency are as follows: L2 ttt  (eq. 6) imp exp 2D timp , texp import time of transiting particles through the NPC, export time

imp  in C ACkK import entrance frequency of transiting particles into the NPC

exp  in N ACkK export entrance frequency

in impC outN in N  ECkkECk exp)1( (eq. 7)

Eimp , Eexp import efficiency of transiting particles through the NPC, export efficiency When the system is at equilibrium ( = 0):

Kimp 2CC P  where  2Ca C ,b  (eq. 8)  btKa exp LAN

E C imp  r where r  N (eq. 9) E exp C C

Figure 21 : Kinetic constants of entrance frequency, transport time and efficiency and the nuclear pore permeability. 56

Therefore, the concentration ratio of transiting particles between the cytoplasm and the nucleus cannot be directly used to reflect the nuclear pore permeability. As shown in eq. 8, the permeability is proportional to the entrance frequency and inversely proportional to the transport time for passive diffusion.

5.2 Calcium-regulated nuclear pore permeability for passive diffusion

Under the normal calcium condition, the transport time of molecules was 1.7 ms, whereas for the calcium depleted condition there was a longer transport time of around 2.8 ms (Fig 10).

The entrance frequencies under calcium depleted conditions were also very low (Fig 14), which suggests a higher barrier on the entrance side of the NPC for small molecules. A longer transport time may be caused by a longer traveling pathway or a higher viscosity or both within the NPC.

The combination of both effects would cause the nuclear pore to become less permeable or even impermeable for passive diffusion. This was evident when the efficiency of calcium depleted conditions reduced drastically compared to the normal calcium conditions. Only 5 % of the small molecules could translocate through the pore when calcium stores were heavily depleted with calcium ATPase pump inhibitor Tg (1 mM) and calcium chelator BAPTA (Fig 13). Although most of the events were abortive events in the later case, two transport times (one- shorter and one-longer) were observed (Figs 12 C-D). Due to the probable high barrier near the entrance some molecules are aborted faster to generate a shorter time, whereas, some other events, traveling to deeper segments of the pore or crossing the pore to reach the other compartment, showed a longer transport time.

Under the high-calcium condition, the nuclear pore allows an easier entrance to the NPC

(about 70 events per second) and a faster diffusion through the NPC (approximately 1.1 ms) for 57 passive diffusion of transiting molecules. Thus a higher permeability is generated for the nuclear pore at high calcium concentrations in the stores. Therefore, the calcium concentration in the stores directly regulates the nuclear pore permeability. There were more complete events than abortive ones in high calcium conditions, raising the transport efficiency up to 60%. Passive diffusion is observed to be affected by altered store calcium concentrations, which is in agreement with previous studies [56, 65, 66]

The determined entrance frequency, transport time and efficiency may also shed a light on the previous conflicting results regarding passive diffusion. As demonstrated in Fig 15, there are about 70 dextran molecules entering a single NPC per second and it takes every molecule about 1 ms to complete the diffusion under the high-calcium condition. On the contrary, under the calcium-depleted condition (1 mM Tg and 10 mM BAPTA), only 1-2 molecules can enter a single pore per second and they only have a 5% probability to finish the transport within 2-4 ms.

The considerable differences in the entrance frequency, transport time and efficiency under the two conditions results in a obvious conclusion. There is an inhibition of passive diffusion induced by the calcium-depleted condition. However, when the calcium in the stores is not dramatically depleted, a contradictory conclusion might be drawn. For example, under the normal-calcium condition and one of the calcium-depleted conditions (0.3 µM Tg and 10 mM

BAPTA), the transport time and the entrance frequency have only slight differences, indicating a negligible effect on the nuclear pore permeability (Fig 15). The difference compared to the highly depleted calcium condition (1 mM Tg and 10 mM BAPTA) is very evident. There was a linear increase in transport time and a linear decrease in transport efficiency and entrance frequency. A false conclusion could be drawn under the circumstances namely that calcium depletion has no effect on passive diffusion. To completely exclude other possibilities, a re- 58 examination of calcium-depletion degree in the calcium stores for the previous conflicting results might be necessary.

5.3 Calcium-regulated nuclear pore permeability for facilitated transport

Facilitated transport is a much more complex mechanism than passive diffusion, as it requires an array of proteins to complete the transport. It involves transport receptors, conjugating proteins and an input of metabolic energy to cross the NPC barrier and reach the other compartment. Imp β recognizes a cargo molecule to form a transport complex either directly or indirectly (i.e., via Importin α) [33, 95, 96]. Imp β promotes the movement of the transport complex through the NPC by a series of transient interactions with the FG-Nups.

Thousands of FG repeats are distributed within a NPC, and Imp β is predicted to have up to ten binding sites on its surface that interact with the FG repeats [97-99]. Indeed, Imp β paves the way for facilitated translocation through the FG-Nups barrier under real-time trafficking conditions. Therefore, Imp β is used for studies of facilitated translocation.

Contradictory results of calcium-depletion effects on facilitated transport were also reported [45]. Some groups suggest changes in facilitated transport occur following cisternal calcium depletion, while some groups have not observed any changes, which lead them to claim that facilitated transport is unaffected by calcium depletion [52, 70]. The conclusions so far are based on ensemble experiments, accounting for the inconsistencies. Thus, with single-molecule experiments, detailed kinetic information for calcium-regulated facilitated nuclear transport provides better insight into the controversial views.

Under the normal calcium condition, the transport time of Imp β molecules was around

3.5 ms, and at high calcium condition the average transport time decreases to be about 1.1 ms. 59

For calcium depleted conditions, the transport times decreased too when compared to normal calcium condition (Fig 17), indicating a change of transport time with any alteration of calcium concentrations in the stores. However, further depletion of calcium did not result in a continuous decrease of transport time for the facilitated transport (Table 4).

At the high and normal calcium conditions, the nuclear pore allowed entrance of almost equal numbers of Imp β molecules to NPC from the cytoplasm, but the entrance frequency did not show the same numbers when the calcium levels were depleted. For one of the calcium depleted conditions the import entrance frequency was low, only 17 events per sec, while the other conditions gave entrance frequencies as high as 60 and 45 events per second (Fig 20). No trend was observed for entrance frequency under various calcium depletion conditions. This could be due to the fact that facilitated transport is a very complicated process involving a variety of proteins. So with altered calcium conditions Imp β may react differently with the associated proteins in the NPC structure and in the vicinity of the NPC. There is a NPC permeability change with the change of calcium concentrations, but this change is not straightforward as during passive diffusion and more research is needed on the permeability change for facilitated transport.

The transport efficiency of Imp β molecules decreased when calcium stores were reduced. At high and normal calcium the transport efficiency for Imp β molecules was almost

50%, but when the stores were depleted, it declined to 10 % (Fig 19). This decrease in transport efficiency of Imp β molecules is quite similar to the decrease in transport efficiency of 10 kDa

Dextran molecules with decreasing calcium concentrations. Thus, the transport efficiency is affected similarly for passive and facilitated transport when calcium is depleted. 60

In conclusion, single molecule methods enable us to measure the transport kinetics that are directly relevant to the nuclear pore permeability for passive diffusion and facilitated transport. A systematic investigation of the calcium-induced changes in nuclear pore permeability clarified the ambiguity associated with previous conflicting results and also unraveled the detailed kinetics of calcium - regulated passive diffusion and facilitated transport.

With more improvements in localization precision and temporal resolution of single molecule imaging, the spatial locations of molecules can be captured and three-dimensional spatial density maps of interaction sites between FG Nups and transiting molecules can be obtained [91].

Further work on three-dimensional distribution of molecules inside the pore and their interactions with the NPC components will give better insights into calcium regulated nuclear transport.

61

VI. REFERENCES

1. Miao, L. and K. Schulten, Transport-Related Structures and Processes of the Nuclear Pore Complex Studied through Molecular Dynamics. Structure, 2009. 17(3): p. 449-459. 2. Tweeny R. Kau, J.C.W.a.P.A.S., Nuclear Transport and Cancer: From Mechnism to Intervention. Nature Reviews Cancer, 2004. 4: p. 106-117. 3. Silver, A.H.C.a.P.A., Nucleocytoplasmic transport of macromolecules. Microbiology and Molecular Biology Reviews, 1997. 61(2): p. 193-211. 4. Terry, L.J., E.B. Shows, and S.R. Wente, Crossing the Nuclear Envelope: Hierarchical Regulation of Nucleocytoplasmic Transport. Science, 2007. 318(5855): p. 1412-1416. 5. Norbert Frey, T.A.M.E.N.O., Decoding calcium signals involved in cardiac growth and function. Nature Medicine, 2000. 6: p. 1221-1227. 6. Mattson, M.P., et al., Calcium signaling in the ER: its role in neuronal plasticity and neurodegenerative disorders. Trends in Neurosciences, 2000. 23(5): p. 222-229. 7. Orrenius, G.E.K.a.S., Calcium signaling and cytotoxicity. Environ Health Perspect., 1999. 107(1): p. 25-35. 8. Pozzan, R.R.T., When calcium goes wrong: genetic alterations of a ubiquitous signaling route. Nature Genetics, 2003. 34: p. 135-141. 9. Callan HG, T.S., Experimental studies on amphibian oocyte nuclei. I. Investigation of the structure of the nuclear membrane by means of the electron microscope. Proc R Soc Lond B Biol Sci, 1950. 137: p. 367-378. 10. Watson, M.L., Further Observations on the Nuclear Envelope of the Animal Cell. J Biophys Biochem Cytol, 1959. 6(2): p. 147-156. 11. Lim, R., U. Aebi, and B. Fahrenkrog, Towards reconciling structure and function in the nuclear pore complex. Histochemistry and Cell Biology, 2008. 129(2): p. 105-116. 12. Birthe Fahrenkrog, E.C.H., Ueli Aebi, and Nelly Panté*, Molecular Architecture of the Yeast Nuclear Pore Complex: Localization of Nsp1p Subcomplexes Journal of Cell Biology, 1998. 143(3): p. 577-588. 13. Beck, M., et al., Nuclear Pore Complex Structure and Dynamics Revealed by Cryoelectron Tomography. Science, 2004. 306(5700): p. 1387-1390. 14. Brohawn, S.G., et al., The Nuclear Pore Complex Has Entered the Atomic Age. 2009. 17(9): p. 1156-1168. 15. Stoffler, D., B. Fahrenkrog, and U. Aebi, The nuclear pore complex: from molecular architecture to functional dynamics. Current Opinion in Cell Biology, 1999. 11(3): p. 391-401. 16. Beck, M., et al., Snapshots of nuclear pore complexes in action captured by cryo-electron tomography. Nature, 2007. 449(7162): p. 611-615. 17. Martin Beck, F.F.r., Mary Ecke, Ju¨rgen M. Plitzko, Frauke Melchior, Gu¨nther Gerisch, Wolfgang Baumeister, Ohad Medalia., Nuclear Pore Complex Structure and Dynamics Revealed by Cryoelectron Tomography. Science, 2004. 306: p. 1387-1390. 18. Köhler, A. and E. Hurt, Gene Regulation by Nucleoporins and Links to Cancer. Molecular Cell, 2010. 38(1): p. 6-15. 19. Aitchison, M.P.R.a.J.D., The Nuclear Pore Complex as a Transport Machine. The Journal of Biological Chemistry, 2001. 276(20): p. 16593-16596. 20. Cronshaw, J.M., et al., Proteomic analysis of the mammalian nuclear pore complex. The Journal of Cell Biology, 2002. 158(5): p. 915-927. 62

21. Fahrenkrog, B., et al., Domain-specific antibodies reveal multiple-site topology of Nup153 within the nuclear pore complex. Journal of Structural Biology, 2002. 140(1-3): p. 254-267. 22. Walther, T.C., et al., The Nup153 is required for nuclear pore basket formation, nuclear pore complex anchoring and import of a subset of nuclear proteins. EMBO J, 2001. 20(20): p. 5703-5714. 23. Rabut, G., V. Doye, and J. Ellenberg, Mapping the dynamic organization of the nuclear pore complex inside single living cells. Nat Cell Biol, 2004. 6(11): p. 1114-1121. 24. Ribbeck, K. and D. Gorlich, Kinetic analysis of translocation through nuclear pore complexes. EMBO J, 2001. 20(6): p. 1320-1330. 25. Xylourgidis, N. and M. Fornerod, Acting Out of Character: Regulatory Roles of Nuclear Pore Complex Proteins. 2009. 17(5): p. 617-625. 26. Terry LJ, S.E., Wente SR., Crossing the nuclear envelope: hierarchical regulation of nucleocytoplasmic transport. Science, 2007. 30: p. 1412-1416. 27. Lucy, F.P. and M.P. Bryce, Mechanisms of Receptor-Mediated Nuclear Import and Nuclear Export. Traffic, 2005. 6(3): p. 187-198. 28. Weis, A.-C.S.a.K., Importin-beta-like nuclear transport receptors. Genome Biology, 2001. 2(6): p. 3008.1-3008.9. 29. Ribbeck, K., et al., NTF2 mediates nuclear import of Ran. EMBO J, 1998. 17(22): p. 6587-6598. 30. Terry, L.J. and S.R. Wente, Flexible Gates: Dynamic Topologies and Functions for FG Nucleoporins in Nucleocytoplasmic Transport. Eukaryotic Cell, 2009. 8(12): p. 1814-1827. 31. Carmody, S.R. and S.R. Wente, mRNA nuclear export at a glance. J Cell Sci, 2009. 122(12): p. 1933-1937. 32. Fried, H. and U. Kutay, Nucleocytoplasmic transport: taking an inventory. Cellular and Molecular Life Sciences, 2003. 60(8): p. 1659-1688. 33. Stewart, M., Molecular mechanism of the nuclear protein import cycle. Nat Rev Mol Cell Biol, 2007. 8(3): p. 195-208. 34. Kuersten, S., M. Ohno, and I.W. Mattaj, Nucleocytoplasmic transport: Ran, beta and beyond. Trends in Cell Biology, 2001. 11(12): p. 497-503. 35. Rout, M.P., et al., Virtual gating and nuclear transport: the hole picture. 2003. 13(12): p. 622- 628. 36. Reiner, P., Translocation Through the Nuclear Pore Complex: Selectivity and Speed by Reduction‐of‐Dimensionality. Traffic, 2005. 6(5): p. 421-427. 37. Ben-Efraim, I. and L. Gerace, Gradient of Increasing Affinity of Importin β for Nucleoporins along the Pathway of Nuclear Import. The Journal of Cell Biology, 2001. 152(2): p. 411-418. 38. Macara, I.G., Transport into and out of the Nucleus. Microbiol. Mol. Biol. Rev., 2001. 65(4): p. 570-594. 39. Fiserova, J. and M.W. Goldberg, Nucleocytoplasmic transport in yeast: a few roles for many actors. Biochemical Society Transactions, 2010. 038(1): p. 273-277. 40. Cook, A., et al., Structural Biology of Nucleocytoplasmic Transport. Annual Review of Biochemistry, 2007. 76(1): p. 647-671. 41. Martin, K., et al., Binding Site Distribution of Nuclear Transport Receptors and Transport Complexes in Single Nuclear Pore Complexes. Traffic, 2009. 10(9): p. 1228-1242. 42. Tran EJ, B.T., Wente SR., SnapShot: nuclear transport. Cell, 2007. 121(2): p. 420. 43. Harvey Lodish, A.B., S. Lawrence Zipursky, Paul Matsudaira, David Baltimore and James Darnell; Freeman & Co, Molecular Cell Biology. 2000. 44. Clapham, D.E., Calcium Signaling. 2007. 131(6): p. 1047-1058. 45. Bootman, M.D., et al., An update on nuclear calcium signalling. J Cell Sci, 2009. 122(14): p. 2337- 2350. 63

46. Gill DL, G.T., Bian J, Short AD, Waldron RT, Rybak SL., Function and organization of the inositol 1,4,5-trisphosphate-sensitive calcium pool. Adv Second Messenger Phosphoprotein Res., 1992. 26: p. 265-308. 47. Erickson ES, M.O., Moore D, Krogmeier JR, Dunn RC., The role of nuclear envelope calcium in modifying nuclear pore complex structure. Can J Physiol Pharmacol. , 2006. 84(3-4): p. 309-318. 48. Lee, M.A.D., Robert C. Clapham, David E. Stehno-Bittel, Lisa, Calcium regulation of nuclear pore permeability. Cell Calcium, 1998. 23(2-3): p. 91-101. 49. Kramer, A., et al., A Pathway Separate from the Central Channel through the Nuclear Pore Complex for Inorganic Ions and Small Macromolecules. Journal of Biological Chemistry, 2007. 282(43): p. 31437-31443. 50. Carmen, P.-T., J. Marisa, and E.C. David, Nuclear calcium and the regulation of the nuclear pore complex. BioEssays, 1997. 19(9): p. 787-792. 51. Klein, C. and A.N. Malviya, Mechanism regulating nuclear calcium signaling. Canadian Journal of Physiology and Pharmacology, 2006. 84: p. 403-422. 52. Gerasimenko, J., et al., Calcium signalling in and around the nuclear envelope. Biochem. Soc. Trans., 2003. 31(Pt 1): p. 76-78. 53. Lim, R.Y.H., et al., Nanomechanical Basis of Selective Gating by the Nuclear Pore Complex. Science, 2007. 318(5850): p. 640-643. 54. Perez-Terzic, C., et al., Structural Plasticity of the Cardiac Nuclear Pore Complex in Response to Regulators of Nuclear Import. Circ Res, 1999. 84(11): p. 1292-1301. 55. Panté, N. and U. Aebi, Molecular Dissection of the Nuclear Pore Complex. Critical Reviews in Biochemistry and Molecular Biology, 1996. 31(2): p. 153-199. 56. Perez-Terzic, C., et al., Conformational States of the Nuclear Pore Complex Induced by Depletion of Nuclear Ca2+ Stores. Science, 1996. 273(5283): p. 1875-1877. 57. Wang, H. and D.E. Clapham, Conformational Changes of the in Situ Nuclear Pore Complex. 1999. 77(1): p. 241-247. 58. Moore-Nichols, D., A. Arnott, and R.C. Dunn, Regulation of Nuclear Pore Complex Conformation by IP3 Receptor Activation. 2002. 83(3): p. 1421-1428. 59. Olivia L Mooren, E.S.E., David Moore-Nichols and Robert C Dunn, Nuclear side conformational changes in the nuclear pore complex following calcium release from the nuclear membrane. Phys. Biol. , 2004. 125-134. 60. Erickson, E., et al., The role of nuclear envelope calcium in modifying nuclear pore complex structure. Can J Physiol Pharmacol. , 2006. 84(3-4): p. 309-318. 61. Malviya, A.N. and P.J. Rogue, "Tell Me Where Is Calcium Bred": Clarifying the Roles of Nuclear Calcium. Cell, 1998. 92(1): p. 17-23. 62. Strubing, C. and D.E. Clapham, Active Nuclear Import and Export Is Independent of Lumenal Ca2+ Stores in Intact Mammalian Cells. J. Gen. Physiol., 1999. 113(2): p. 239-248. 63. Huang, N.-P., et al., Towards monitoring transport of single cargos across individual nuclear pore complexes by time-lapse atomic force microscopy. Journal of Structural Biology, 2010. 171(2): p. 154-62. 64. Paulillo, S.M., et al., Changes in Nucleoporin Domain Topology in Response to Chemical Effectors. Journal of Molecular Biology, 2006. 363(1): p. 39-50. 65. Stehno-Bittel, L., C. Perez-Terzic, and D.E. Clapham, Diffusion Across the Nuclear Envelope Inhibited by Depletion of the Nuclear Ca2+ Store. Science, 1995. 270(5243): p. 1835-1838. 66. Greber, U.F. and L. Gerace, Depletion of calcium from the lumen of endoplasmic reticulum reversibly inhibits passive diffusion and signal-mediated transport into the nucleus. The Journal of Cell Biology, 1995. 128(1): p. 5-14. 64

67. U F Greber, A.S., and L Gerace, A major glycoprotein of the nuclear pore complex is a membrane- spanning polypeptide with a large lumenal domain and a small cytoplasmic tail. EMBO J., 1990. 9(5): p. 1495-1502. 68. Greber, U.F. and L. Gerace, Nuclear protein import is inhibited by an antibody to a lumenal epitope of a nuclear pore complex glycoprotein. The Journal of Cell Biology, 1992. 116(1): p. 15- 30. 69. Gensburger, C., et al., In vivo nuclear Ca2+-ATPase phosphorylation triggers intermediate size molecular transport to the nucleus. Biochemical and Biophysical Research Communications, 2003. 303(4): p. 1225-1228. 70. Wei, X., et al., Real-Time Imaging of Nuclear Permeation by EGFP in Single Intact Cells. Biophysical Journal, 2003. 84(2): p. 1317-1327. 71. Enss, K., et al., Passive Transport of Macromolecules through Xenopus laevis Nuclear Envelope. Journal of Membrane Biology, 2003. 196(3): p. 147-155. 72. O'Brien, E.M., et al., Hormonal Regulation of Nuclear Permeability. Journal of Biological Chemistry, 2007. 282(6): p. 4210-4217. 73. Ha, T., Single-molecule fluorescence methods for the study of nucleic acids. Current Opinion in Structural Biology, 2001. 11(3): p. 287-292. 74. Zlatanova, J. and K. van Holde, Single-Molecule Biology: What Is It and How Does It Work? 2006. 24(3): p. 317-329. 75. Paul R. Selvin, T.H., Single-Molecule Techniques. 2008, New York: John Inglis. 76. Joo, C., et al., Advances in Single-Molecule Fluorescence Methods for Molecular Biology. Annual Review of Biochemistry, 2008. 77(1): p. 51-76. 77. Zlatanova, J., et al., Chromatin structure and dynamics: lessons from single molecule approaches, in New Comprehensive Biochemistry. 2004, Elsevier. p. 369-396. 78. Greve, M.L.B.S.H.L.G.H.L.J.Z.B.G.d.G.J., Unfolding individual nucleosomes by stretching single chromatin fibers with optical tweezers. Nat Struct Biol. 2001 2001. 8(7): p. 606-610. 79. Allemand, J.F., et al., Stretched and overwound DNA forms a Pauling-like structure with exposed bases. Proceedings of the National Academy of Sciences of the United States of America, 1998. 95(24): p. 14152-14157. 80. Herbert, K.M., et al., Sequence-Resolved Detection of Pausing by Single RNA Polymerase Molecules. 2006. 125(6): p. 1083-1094. 81. Lakadamyali, M., et al., Visualizing infection of individual influenza viruses. Proceedings of the National Academy of Sciences of the United States of America, 2003. 100(16): p. 9280-9285. 82. Yang, W., J. Gelles, and S.M. Musser, Imaging of single-molecule translocation through nuclear pore complexes. Proceedings of the National Academy of Sciences of the United States of America, 2004. 101(35): p. 12887-12892. 83. Kubitscheck, U., et al., Nuclear transport of single molecules. The Journal of Cell Biology, 2005. 168(2): p. 233-243. 84. Yildiz, A., et al., Myosin V Walks Hand-Over-Hand: Single Fluorophore Imaging with 1.5-nm Localization. Science, 2003. 300(5628): p. 2061-2065. 85. Yildiz, A., et al., Kinesin Walks Hand-Over-Hand. Science, 2004. 303(5658): p. 676-678. 86. Babcock, H.P., C. Chen, and X. Zhuang, Using Single-Particle Tracking to Study Nuclear Trafficking of Viral Genes. Biophysical Journal, 2004. 87(4): p. 2749-2758. 87. Dange, T., et al., Autonomy and robustness of translocation through the nuclear pore complex: a single-molecule study. The Journal of Cell Biology, 2008. 183(1): p. 77-86. 88. Yang, W. and S.M. Musser, Nuclear import time and transport efficiency depend on importin β concentration. The Journal of Cell Biology, 2006. 174(7): p. 951-961. 65

89. Yang, W. and S.M. Musser, Visualizing single molecules interacting with nuclear pore complexes by narrow-field epifluorescence microscopy. Methods, 2006. 39(4): p. 316-328. 90. Sun, C., et al., Single-molecule measurements of importin α/cargo complex dissociation at the nuclear pore. Proceedings of the National Academy of Sciences, 2008. 105(25): p. 8613-8618. 91. Ma, J. and W. Yang, Three-dimensional distribution of transient interactions in the nuclear pore complex obtained from single-molecule snapshots. Proceedings of the National Academy of Sciences, 2010. 107(16): p. 7305-7310. 92. D Görlich, N.P., U Kutay, U Aebi, and F R Bischoff, Identification of different roles for RanGDP and RanGTP in nuclear protein import. EMBO J., 1996. 15(20): p. 5584–5594. 93. Rexach, M. and G. Blobel, Protein import into nuclei: association and dissociation reactions involving transport substrate, transport factors, and nucleoporins. Cell, 1995. 83(5): p. 683-692. 94. OLE THASTRUP, P.J.C., BJORN K. DROBAK, MICHAEL R. HANLEY, AND ALAN P. DAWSON, Thapsigargin, a tumor promoter, discharges intracellular Ca2+ stores by specific inhibition of the endoplasmic reticulum Ca2+-ATPase. Proc. Natl. Acad. Sci., 1990. 87: p. 2466-2470. 95. Kylie, M.W. and A.J. David, Importins and Beyond: Non-Conventional Nuclear Transport Mechanisms. Traffic, 2009. 10(9): p. 1188-1198. 96. Palmeri, D. and M.H. Malim, Importin beta Can Mediate the Nuclear Import of an Arginine-Rich Nuclear Localization Signal in the Absence of Importin alpha. Mol. Cell. Biol., 1999. 19(2): p. 1218-1225. 97. Denning, D.P., et al., Disorder in the nuclear pore complex: The FG repeat regions of nucleoporins are natively unfolded. Proceedings of the National Academy of Sciences of the United States of America, 2003. 100(5): p. 2450-2455. 98. Isgro, T.A. and K. Schulten, Binding Dynamics of Isolated Nucleoporin Repeat Regions to Importin-[beta]. Structure, 2005. 13(12): p. 1869-1879. 99. Bednenko, J., G. Cingolani, and L. Gerace, Importin β contains a COOH-terminal nucleoporin binding region important for nuclear transport. The Journal of Cell Biology, 2003. 162(3): p. 391- 401.