Roles of TACC related protein, mia1p in MTOCs and microtubule dynamics in schizosaccharomyces pombe 2

pombe cells, other than the spindle pole body SPB which is equivalent to the mammalian centrosome, two other transient, cell-cycle-regulated nucleation sites defined here as non-centros

is composed of α/β tubulin heterodimers that are longitudinally linked However microtubules are not static structures but are highly dynamic, undergoing repeated transitions between growth and shrinkage, a phenomenon called dynamic instability (for

review, see Dammermann et al., 2003) In an in vitro situation, during polymerizing,

growing state, α/β-tubulin subunits are added to both ends of the microtubule with different rates: the slow growing end terminated by α-tubulin is defined as “minus end”, while the fast growing end capped by β-tubulin is termed “plus end” (for review, see

Dammermann et al., 2003) As tubulin heterodimers bind to the growing ends of

microtubules, β-tubulin guanosine triphosphate (GTP) within the heterodimer subsequently hydrolyzes to guanosine diphosphate (GDP), leaving GDP bound form of tubulin subunits in the lattice Hydrolysis of GTP causes conformation changes in the tubulin subunits but the resulting curved protofilaments are stabilized by the lateral bonds within the microtubule wall, especially in the vicinity of the stable cap of GTP-containing

subunits at microtubule plus end During shrinking state, the GTP cap is lost from the plus end of microtubule and the unstable curved protofiliments peel back from the microtubule wall Eventually, the protofilaments disassemble into free tubulin heterodimers (for text book, see Molecular Biology of the cell, Third Edition) The dynamic instability of microtubules is shown on the following cartoon (for review, see Wiese and Zheng, 2006)

However in an in vivo environment lacking a relatively high tubulin

concentration, microtubule nucleation requires another member of the tubulin family, tubulin γ-tubulin is found in a large protein complex called γ-tubulin ring complex (γ-TuRC) that is present both at the centrosome and non-centrosomal microtubule

γ-nucleation sites to promote assembly of microtubules (Raynaud-Messina and Merdes, 2007)

1.1.2 Centrosome

The centrosome is thought to nucleate the majority of microtubules in many animal cells In the fluorescence microscopy analyses, the microtubules are seen in greatest density around the nucleus and radiate out into the cell periphery during interphase This ordered microtubule network is organized by one key player—the centrosome Centrosome consists of a pair of centrioles surrounded by an amorphous cloud of pericentriolar material (Bornens, 2002) Functions of centrioles within the centrosome are still poorly understood, although centrioles are thought to serve as the organizer of the pericentriolar material based on the observation of pericentriolar material

dispersion upon microinjection of antibody to disassemble the centrioles (Bobinnec et al.,

1998) Nevertheless, there is now little doubt that γ-tubulin ring complexes (γ-TuRCs), identified within the pericentriolar material, are responsible for nucleating microtubules

(Bobinnec et al., 1998; Wiese and Zheng, 2006) The detailed function of γ-TuRC will be

discussed later in this thesis

1.1.3 Non-centrosomal microtubule

Non-centrosomal microtubules generated by centrosome-independent mechanisms are also found in neurons and epithelial cells as well as skeletal muscle cells (Bartolini and Gundersen, 2006)

How the non-centrosomal microtubules are formed is poorly understood Recent data from studies of non-centrosomal microtubules propose that there are several general mechanisms that cells might employ to nucleate non-centrosomal microtubules Firstly, existing microtubules may be released from the centrosome probably through action of some severing proteins such as Katanin (for review, see Bartolini and Gundersen, 2006; McNally and Vale, 1993) Secondly, microtubules are nucleated from the free γ-TuRCs present in the cytoplasmic form (for review, see Bartolini and Gundersen, 2006) Thirdly,

microtubules can be nucleated from distinct non-centrosomal sites such as trans- Golgi network in human cells (Efimov et al., 2007) Forth, existing microtubules could break

into two parts to generate free microtubule under mechanical stress (for review, see Bartolini and Gundersen, 2006)

1.1.4 Schizosaccharomyces pombe (S pombe) as a model for studying MT dynamics

The unicellular model organism, fission yeast Schizosaccharomyces pombe (S pombe) is an attractive model system to investigate various aspects of microtubule

dynamics Straightforward genetic analyses and a fully sequenced and annotated genome

(Wood et al., 2002) are useful in dissecting molecular mechanisms of microtubule

organization A relatively large cell size allows detailed dynamic observation of cytoskeletal filaments and cellular components tagged with fluorescent proteins Importantly, fission yeast cells exhibit simple but distinct types of microtubule arrays depending on the cell cycle stage

So far four fission yeast tubulin genes have been identified: γ tubulin (gtb1/tug1),

α tubulin (nda2, atb2) and a β tubulin (nda3) (Yanagida, 1987) Among these,

α-tubulin and β-α-tubulin are components of microtubules, while γ-α-tubulin, identified within the microtubule organizing centers, acts as a microtubule nucleating template (for review, see Hagan, 1998)

1.2MTOCs and microtubule cytoskeleton in Schizosaccharomyces pombe (S pombe)

The vegetative cell cycle of S pombe consists of interphase (including G1, S and

G2 phases) and mitosis To perform their functions at different cell stage, microtubules

are organized into complex arrays by MTOCs In S pombe cells, other than the spindle

pole body (SPB) which is equivalent to the mammalian centrosome, two other transient, cell-cycle-regulated nucleation sites (defined here as non-centrosomal MTOCs) are known: the interphase MTOCs (iMTOCs) and the equatorial MTOC (eMTOC) (Hagan

and Petersen, 2000; Tran et al., 2001)

1.2.1 Microtubule Nucleating sites—MTOCs in S pombe

1.2.1.1 Spindle pole body (SPB)

The fission yeast spindle pole bodies (SPBs) are functionally analogous to centrosomes and undergo a duplication and separation cycle, correlated with the cell

division cycle Like the centrosome of vertebrate cells, the SPB of S pombe spends most

of interphase in the cytoplasm, immediately next to the nuclear envelope (NE) (Ding et al., 1997; Uzawa et al., 2004) Currently, it is still a controversial issue about the

duplicating time of SPBs: Some researchers think that it occurs in the late G2 phase as

shown in the following cartoon (from Ding et al., 1997) Another opinion is that SPBs are duplicated at the G1/S boundary and matured in the G2 phase (Uzawa et al., 2004)

Unlike other vertebrate cells undergoing open mitosis in which the NE disassembles in early prophase, fission yeast undergoes “closed mitosis”: The NE remains intact throughout the cell cycle To ensure the access of microtubules to the hereditary material,

a portion of the NE underlying the SPB pair breaks down to form an opening called

“fenestra” once the cell enters mitosis Then the duplicated SPBs settle into fenestra to nucleate intranuclear microtubules During the elongation of the spindle, SPBs are always localized at the leading edge of the NE As anaphase proceeds, the nuclear fenestrae close, and the SPBs are extruded back into the cytoplasm to nucleate interphase microtubules The summary of dynamics of the NE and the SPBs through out the cell

cycle is shown in the following cartoon (Ding et al., 1997)

Diagram summarizing dynamics of the NE and the SPBs throughout the cell cycle

The SPB (shaded ellipse with line) is associated with an appendage (small solid ellipse) and lies close to the NE Late in G2 phase, the SPB duplicates and matures, daughter SPBs are connected by a bridge derived from the appendage Upon mitotic entry, the NE invaginates, perforates to form fenestra and the SPB settle into it The two halves of the structure separate as the spindle forms, such that each SPB occupies its own fenestra At the end of mitosis, the fenestrae close and extrude the SPB back into the cytoplasm for the next interphase

1.2.1.2 Non-centrosomal MTOCs

Compared to the SPBs which exist throughout the cell cycle, the iMTOCs and the eMTOC are assembled at different cell cycle stages: The eMTOC localizes to the cell

center to nucleate the post anaphase array (PAA) at the end of mitosis (Venkatram et al.,

2005) On the other hand, the iMTOCs are only present in interphase cells and disassemble once the cells enter mitosis So far, all components found in the eMTOC are also present in the iMTOCs It is believed that disassembly of the eMTOC is synchronous

with the establishment of the iMTOCs (Zimmerman et al., 2004a)

1.2.1.2.1 Interphase microtubule organizing centers (iMTOCs)

The definition of iMTOCs is still largely controversial to date Some researchers refer to iMTOCs as “satellites” of γ-tubulin complex proteins (Janson et al., 2005) In my opinion, the iMTOCs are the sites on the NE where interphase microtubules are nucleated

It is hard to observe the iMTOCs structures in live cells at steady state due to low fluorescence intensity and distribution of γ-TuRC components along microtubule bundles However, after drug-induced microtubule depolymerization, γ-TuRC components aggregate at microtubule nucleation sites and the iMTOCs can be observed

as several dots around the NE by tagging the components of γ-TuRC with green fluorescent protein (GFP) Repolymerization of microtubules will be initiated from such dots by washing out the depolymerizing agent, demonstrating the microtubule-nucleating capability of the iMTOCs

It is known that the establishment of the iMTOCs is linked to the disassembly of

the eMTOC (Zimmerman et al., 2004a), but the precise origin of the iMTOCs remains

elusive

1.2.1.2.2 Equatorial microtubule organizing center (eMTOC)

At the end of anaphase, the mitotic spindle breaks down and microtubules originate from the middle of the cell to create the PAA (Hagan and Hyams, 1988) The PAA is nucleated from a distinct MTOC called the eMTOC In fluorescence microscope analyses, the eMTOC appear as a ring structure co-localizing with the actomyosin ring in the middle of the dividing cell

Assembly of the eMTOC requires the activity of a GTPase signaling cascade known as the septation initiation network (SIN) that regulates the onset of cytokenesis

(Heitz et al., 2001) In addition, integrity of the eMTOC relies on the integrity of the

F-actin but not the microtubules: the dispersal of the eMTOC in the absence of the actomyosin ring indicates the essential role of actomyosin ring in eMTOC formation

(Heitz et al., 2001) Recent studies found that the eMTOC breaks down into small pieces defined as satellites that can move along the microtubules (Zimmerman et al., 2004a;

Sawin and Tran, 2006)

During the eMTOC-breakdown process, the DnaJ domain protein, Rsp1p, functions in disassembly of the eMTOC, and possibly in assembly of the iMTOCs,

probably through interacting with the cytoplasmic hsp70 protein, Ssa1p (Zimmerman et al., 2004a) It’s believed that Rsp1p may stimulate the ATPase activity of Ssa1p, which

then exerts an ATP-dependent conformational change on its substrate, causing the

protein-protein interaction to weaken and resulting in the breakdown of the eMTOC

(Zimmerman et al., 2004a) The establishment of the iMTOCs is believed to proceed

synchronously with the disassembly of the eMTOC since γ-TuRC components were found to move from eMTOC to the iMTOCs as satellites when the eMTOC ring

constricts (Zimmerman et al., 2004a) Failure to disassemble the eMTOC in the rsp1-1

mutant leads to a defect in the organization of interphase cytoplasmic microtubules

(Zimmerman et al., 2004a)

1.2.1.2.3 γ-TuRC satellites

In addition to the MTOCs mentioned above, when fused with GFP, the γ-TuRC components are also seen as weakly fluorescent dots traveling along the interphase

microtubules in a bi-directional manner (Zimmerman et al., 2004a) Such satellites are

capable of nucleating microtubules on the preexisted microtubule in interphase cells

microtubule nucleating capacity on its own in vitro [(Oegema et al., 1999)

In addition to γ-tubulin, identified as the key component of the MTOCs, two other highly conserved proteins are found to be present in the γ-TuRC in all eukaryotes:

Spc97p and Spc98p in the budding yeast (Saccharomyces cerevisiae) or Gcp2p and Gcp3p in human (Knop and Schiebel, 1997; Zimmerman et al., 2004b) Other

components of γ-TuRC vary in different species

In fission yeast, the components of γ-TuRC include Tug1/ Gtb1 (γ-tubulin) (Horio

et al., 1991) together with Alp4p and Alp6p which are homologs of Spc97p and Spc98p respectively But unlike the S cerevisiae γ-tubulin complex, additional members of the γ- TuRC, Alp16p and Gfh1p, have been identified in S pombe Database searches revealed

that they share sequence similarity with regions of hGCP6 and hGCP4, respectively

(Fujita et al., 2002; Venkatram et al., 2004) These γ-TuRC components localize to the

SPBs both in interphase and mitotic cells and also on the iMTOCs and eMTOC (Vardy

and Toda, 2000; Fujita et al., 2002; Venkatram et al., 2004)

Among γ-TuRC components, the γ-tubulin, Alp4p and Alp6p are required for cell viability and are essential for various aspects of microtubule functions Since cells lacking such components die due to defects in formation of mitotic bipolar spindles

(Tange et al., 2004), their functions in interphase microtubule organization are largely

founded on phenotypes of temperature-sensitive mutants (Vardy and Toda, 2000) Another two γ-TuRC members—Alp16p and Ghf1p are not essential for cell viability They are required for the formation of normal interphase microtubules but have little

effect on the function of the mitotic spindle (Fujita et al., 2002; Venkatram et al., 2004)

This suggests that different members of the γ-TuRC play specific roles in microtubule organization However, defects in any of the γ-TuRC components typically exhibit

similar interphase phenotypes: reduced number of microtubule bundles; the microtubules

do not stop growing when their plus ends touch the cell tips, instead, they continue to

grow and produce longer microtubules which curve around the cell tips (Fujita et al., 2002; Samejima et al., 2005)

How do γ-TuRC components in the minus ends of microtubules contribute to microtubule dynamic is yet unclear It is possible that in addition to the factors such as Mal3p or Tip1p that directly binding to microtubule plus ends to prevent pre-matured catastrophe at cell cortex until microtubules touch cell tips (Busch and Brunner, 2004), other factors that regulating microtubule plus-end dynamic need to be loaded at the microtubule minus ends during nucleation: One sample is kinesin Teap2p that is loaded onto the microtubules in close proximity to the nucleus and then travels using its intrinsic

motor activity primarily at the tips of polymerizing microtubules (Browning et al., 2003;

Sawin and Tran, 2006) Also, the transforming acidic coiled-coil (TACC)-related protein, Mia1p (or Alp7p), localizes at the SPB to recruit Alp14p (homologue of XMAP215p) to

stabilize microtubules (Sato et al., 2004).Alternatively, such changes could be due to the disturbed balance between plus ends of microtubules and microtubule stabilizing proteins and free tubulin subunits Further investigations of γ-TuRC components should provide new insights to this issue

1.2.2.2 Mto1p and Mto2p

Recently, two other novel proteins have been identified as γ-TuRC components in fission yeast—Mto1p (also known as Mod20p and Mbo1p) and Mto2p These two proteins physically interact with each other and can co-immunoprecipitate with γ-tubulin

They are non-essential genes and neither of them are required for assembly of mitotic

spindle (Samejima et al., 2005) However, interphase microtubules are longer and curved around the cell tips in both mto1Δ and mto2Δ cells, a typical phenotype of other γ-TuRC

mutants The defects in nucleating cytoplasmic microtubules from non-spindle pole body MTOCs—iMTOCs suggest that Mto1p and Mto2p function by recruiting the γ-tubulin

complex to non-spindle pole body MTOCs for microtubule nucleation (Sawin et al., 2004) By database searching, several proteins (such as D melanogaster centrosomin)

sharing similar sequence to Mto1p have been found in filamentous fungi and higher eukaryotes suggesting that a general mechanism for the organization of noncentrosomal

MTOCs is conserved in eukaryotic cells (Sawin et al., 2004; Venkatram et al., 2005)

1.2.3 Organization of microtubule bundles

To organize an ordered microtubule network, a wide array of components is required: Firstly, microtubule nucleators, MTOCs, need to maintain the right amount of microtubules and localize them at precise sites Secondly, crosslinkers are required to bundle microtubules Thirdly, motor proteins mediate microtubule sliding within the microtubule bundles to facilitate the maintenance of microtubule polarity (Carazo-Salas and Nurse, 2007)

In interphase S pombe cells, in addition to microtubules nucleated by the SPBs

and the iMTOCs around the NE, new microtubules are also generated from the satellites

present on the preexisting microtubules (Janson et al., 2007) These microtubules are

transported to the overlap region around the NE where they are stabilized This process functions to maintain the overall antiparallel microtubule arrangement (Sawin and Tran,

2006) Recent work in microtubule dynamics found that at least two proteins are involved

in this process One of them is the minus-end-directed protein—Klp2p

1.2.3.1 Minus-end-directed motor protein—Klp2p

Klp2p is a kinesin of the KAR3 subfamily in fission yeast In mitotic cells, it localizes to kinetochores to regulate elongation of the anaphase spindle and to control appropriate disassembly of the spindle at the completion of mitosis (Troxell, 2001) During interphase, Klp2p localizes as numerous dots which accumulate only at plus ends

of both existing mother microtubules and newly nucleated daughter microtubules but is absent from the minus ends of individual microtubules, suggesting that sliding forces

between overlapping microtubules are generated only at microtubule plus ends (Janson et al., 2007), thus the plus end of a newly nucleated microtubule serves as a cargo of Klp2p

to be delivered to the minus end of mother microtubule

In the absence of Klp2p, sliding of newly nucleated microtubules on pre-existing microtubules is compromised, leading to defects in focusing of microtubules at the

overlapping regions (Carazo-Salas et al., 2005) This suggests that Klp2p functions as a

microtubule slider to transport newly nucleated microtubules to the overlapping region, thus contributing to maintenance of the antiparallel linear microtubule arrays in

interphase cells

1.2.3.2 Bundling protein—Ase1p

Another protein which functions in organizing antiparallel microtubule arrangement is Ase1p The yeast Ase1p belongs to the conserved ASE1/PRC1/MAP65

family of microtubule bundling proteins found at the mitotic spindle midzone (Chan et al., 1999) Ase1p functions in both interphase and mitosis in S pombe cells During

mitosis, it localizes to mitotic spindle midzone where the plus ends of microtubule

overlap to stabilize bipolar spindle and prevents it from collapsing (Loiodice et al., 2005)

In interphase cells, Ase1p localizes at the regions where minus ends of microtubules overlap, including regions around the MTOCs at the NE and overlapping regions of mother-daughter microtubules when daughter microtubule is nucleated by the γ-TuRC satellites on the mother microtubules In ase1Δ mutants, interphase microtubules are

disorganized and fail to form overlapping antiparallel microtubule bundles (Loiodice et al., 2005), suggesting that the role of Ase1p in S pombe cells is to act as a microtubule

bundler

1.2.3.3 Model of maintaining ordered microtubules

MTOCs concentrate microtubule nucleation, attachment and bundling factors which coordinate each other in effectively organizing interphase microtubules into ordered anti-parallel linear arrays Recent studies in MTOCs discover more and more components of the γ-TuRC Their functions in organizing microtubules have been characterized, providing more clues and leading to form a model of organizing the microtubule bundles (Carazo-Salas and Nurse, 2007) Data published so far supported a model in which interplay between bundling proteins—Ase1p and the minus-end directed motor protein—Klp2p are sufficient to generate bipolar antiparallel microtubule array (Sawin and Tran, 2006)

According to this model, once the γ-TuRC satellite has nucleated a new daughter microtubule on a pre-existing mother microtubule, Ase1p and Klp2p will ensure that mother and daughter microtubules are further organized into functional patterns The inherent ability of Ase1p allows it to bind only at microtubule antiparallel regions but makes it absent from other regions on microtubule arrays By forming oligomers, Ase1p

is capable of bringing two or more microtubule binding domains together, thus to bundle

and stabilize the antiparallel arrangement of daughter-mother microtubules (Janson et al.,

2007) Bundling activity of Ase1p is dynamic in order to allow microtubules remodeling and sliding Additionally, Klp2p is recruited to the growing plus-end tip of the daughter microtubule, pulling it to move toward the cell center along mother microtubules The pulling forces generated by Klp2p effectively slide the daughter and mother microtubules relative to each other (Sawin and Tran, 2006) However sliding forces are negatively affected by the Ase1p bundling Thus, as daughter microtubule continues to grow, more Ase1p molecules are recruited to the increasing daughter-mother microtubule overlapping region, causing sliding to attenuate No further sliding occurs when Klp2p reaches the end of the mother microtubule In this way, microtubules are organized into

an ordered and functional array with minus-ends bundled together at the cell center and plus-ends facing toward the cell tips Model of maintaining ordered microtubules (from Sawin and Tran, 2006) is shown in the following cartoon