Microtubule capture: a concerted effort

Kinetochores direct attachment of chromosomes to microtubules of the mitotic spindle during cell division. Three recent studies in Cell, including one in this issue, reveal important new roles for two kinetochore protein complexes-Ndc80 and INCENP-Survivin-in establishing the correct attachment of chromosomes to spindle microtubules (Cheeseman et al., 2006, DeLuca et al., 2006 and Sandall et al., 2006).     

Organization of microtubules in stabilized meristematic plant cells revealed by a rat monoclonal antibody reacting only with the tyrosinated form of alpha-tubulin

A rat monoclonal antibody against yeast tubulin (clone YL 1/2; Kilmartin et al., 1982) that reacts specifically with mammalian alpha-tubulin carrying a carboxyterminal tyrosine residue (Wehland et al., 1983) was used to localize microtubules in plant cells derived from onion root apices (Allium cepa L.). YL 1/2 reacted with all types of microtubular arrays known to occur in higher plant meristematic cells such as interphase cortical microtubules, pre-prophase bands, the mitotic spindle and phragmoplast microtubules. The specific labeling of microtubules in isolated cells from onion root tips by YL 1/2 indicates that plant cells like animal cells contain tubulin tyrosine ligase, the enzyme which posttranslationally modifies alpha-tubulin. This enzyme could be involved in the dynamic regulation of microtubular arrays in all eukaryotic cells.     

Conformations of kinesin: solution vs. crystal structures and interactions with microtubules

Recently, the molecular structures of monomeric and dimeric kinesin constructs in complex with ADP have been determined by X-ray crystallography (Kull et al. 1996; Kozielski et al. 1997 a; Sack et al. 1997). The “motor” or “head” domains have almost identical conformations in the known crystal structures, yet the kinesin dimer is asymmetric: the orientation of the two heads relative to the coiled-coil formed by their neck regions is different. We used small angle solution scattering of kinesin constructs and microtubules decorated with kinesin in order to find out whether these crystal structures are of relevance for kinesin's structure under natural conditions and for its interaction with microtubules. Our preliminary results indicate that the crystal structures of monomeric and dimeric kinesin are similar to their structures in solution, though in solution the center-of-mass distance between the motor domains of the dimer could be slightly greater. The crystal structure of dimeric kinesin can be interpreted as representing two equivalent conformations. Transitions between these or very similar conformational states may occur in solution. Binding of kinesin to microtubules has conformational effects on both, the kinesin and the microtubule. Solution scattering of kinesin decorated microtubules reveals a peak in intensity that is characteristic for the B-surface lattice and that can be used to monitor the axial repeat of the microtubules under various conditions. In decoration experiments, dimeric kinesin dissociates, at least partly, leading to a stoichiometry of 1:1 (one kinesin head per tubulin dimer; Thormählen et al. 1998 a) in contrast to the stoichiometry of 2:1 reported for dimeric ncd. This discrepancy is possibly due to the effect of steric hindrance between kinesin dimers on adjacent binding sites.     

Polymerization of tubulin in vivo: direct evidence for assembly onto microtubule ends and from centrosomes

Microtubule assembly in vivo was studied by hapten-mediated immunocytochemistry. Tubulin was derivatized with dichlorotriazinylaminofluorescein (DTAF) and microinjected into living, interphase mammalian cells. Sites of incorporation were determined at the level of individual microtubules by double-label immunofluorescence. The haptenized tubulin was localized by an anti-fluorescein antibody and a second antibody conjugated with fluorescein. Total microtubules were identified by anti-tubulin and a secondary antibody conjugated with rhodamine. Contrary to recent studies (Salmon, E. D., et al., 1984, J. Cell Biol., 99:2165-2174; Saxton, W. M., et al., 1984, J. Cell Biol., 99:2175-2186) which suggest that tubulin incorporates all along the length of microtubules in vivo, we found that microtubule assembly in interphase cells was in vivo, as in vitro, an end-mediated process. Microtubules that radiated out toward the cell periphery incorporated the DTAF-tubulin solely at their distal, that is, their plus ends. We also found that a proportion of the microtubules connected to the centrosomes incorporated the DTAF-tubulin along their entire length, which suggests that the centrosome can nucleate the formation of new microtubules.     

Microtubules with 15 subunits in cockroach epidermal cells

Since Ledbetter and Porter (1964) described the 13 subunits which are visible in cross sections of negatively stained plant microtubules, subsequent observations have generally confirmed this number. By using Mizuhira's fixative composed of tannic acid and glutaraldehyde, it is easyto demonstrate the subunits of microtublules without optical reinforcement Cytoplasmic microtubules and sperm axonemes, fixed with Mizuhira's fixtive, similarly show 13 subunits (Mizuhira's and Futaesaku, 1971, 1972; Futaesaky et al., 1972; Tilney et al., 1973). This paper will describe a particular type of microtubule in insect epidermal cells fixed with the above fixative. The number of the subunits is found to be 15 in tranverse sections.     

Direct interaction of Gas11 with microtubules: implications for the dynein regulatory complex

We previously described the Trypanin family of cytoskeleton-associated proteins that have been implicated in dynein regulation [Hill et al., J Biol Chem2000; 275(50):39369-39378; Hutchings et al., J Cell Biol2002;156(5):867-877; Rupp and Porter, J Cell Biol2003;162(1):47-57]. Trypanin from T. brucei is part of an evolutionarily conserved dynein regulatory system that is required for regulation of flagellar beat. In C. reinhardtii, the trypanin homologue (PF2) is part of an axonemal 'dynein regulatory complex' (DRC) that functions as a reversible inhibitor of axonemal dynein [Piperno et al., J Cell Biol1992;118(6):1455-1463; Gardner et al., J Cell Biol1994;127(5):1311-1325]. The DRC consists of an estimated seven polypeptides that are tightly associated with axonemal microtubules. Association with the axoneme is critical for DRC function, but the mechanism by which it attaches to the microtubule lattice is completely unknown. We demonstrate that Gas11, the mammalian trypanin/PF2 homologue, associates with microtubules in vitro and in vivo. Deletion analyses identified a novel microtubule-binding domain (GMAD) and a distinct region (IMAD) that attenuates Gas11-microtubule interactions. Using single-particle binding assays, we demonstrate that Gas11 directly binds microtubules and that the IMAD attenuates the interaction between GMAD and the microtubule. IMAD is able to function in either a cis- or trans-orientation with GMAD. The discovery that Gas11 provides a direct linkage to microtubules provides new mechanistic insight into the structural features of the dynein-regulatory complex.     

Heterotrimeric G proteins: new tricks for an old dog

Heterotrimeric G proteins are well known for their function in signal transduction downstream of seven transmembrane receptors. More recently, however, genetic analysis in C. elegans and in Drosophila has revealed a second, essential function of these molecules in positioning the mitotic spindle and attaching microtubules to the cell cortex. Five new publications in Cell (Afshar et al., 2004; Du and Macara, 2004 [this issue of Cell]; Hess et al., 2004), Developmental Cell (Martin-McCaffrey et al., 2004), and Current Biology (Couwenbergs et al., 2004) show that this function is conserved in vertebrates and--like the classical pathway--involves cycling of G proteins between GDP and GTP bound conformations.     

Tubulin folding cofactor D is a microtubule destabilizing protein

A rapid switch between growth and shrinkage at microtubule ends is fundamental for many cellular processes. The main structural components of microtubules, the alphabeta-tubulin heterodimers, are generated through a complex folding process where GTP hydrolysis [Fontalba et al. (1993) J. Cell Sci. 106, 627-632] and a series of molecular chaperones are required [Sternlicht et al. (1993) Proc. Natl. Acad. Sci. USA 90, 9422-9426; Campo et al. (1994) FEBS Lett. 353, 162-166; Lewis et al. (1996) J. Cell Biol. 132, 1-4; Lewis et al. (1997) Trends Cell Biol. 7, 479-484; Tian et al. (1997) J. Cell Biol. 138, 821-823]. Although the participation of the cofactor proteins along the tubulin folding route has been well established in vitro, there is also evidence that these protein cofactors might contribute to diverse microtubule processes in vivo [Schwahn et al. (1998) Nature Genet. 19, 327-332; Hirata et al. (1998) EMBO J. 17, 658-666; Fanarraga et al. (1999) Cell Motil. Cytoskel. 43, 243-254]. Microtubule dynamics, crucial during mitosis, cellular motility and intracellular transport processes, are known to be regulated by at least four known microtubule-destabilizing proteins. OP18/Stathmin and XKCM1 are microtubule catastrophe-inducing factors operating through different mechanisms [Waters and Salmon (1996) Curr. Biol. 6, 361-363; McNally (1999) Curr. Biol. 9, R274-R276]. Here we show that the tubulin folding cofactor D, although it does not co-polymerize with microtubules either in vivo or in vitro, modulates microtubule dynamics by sequestering beta-tubulin from GTP-bound alphabeta-heterodimers.     

Nucleation of macrotubules on double-walled microtubule seeds

It is known that histone H1 is able to cause the formation of double-walled microtubules from microtubule protein. Now, we demonstrate that in dependence on the mass ratio H1/microtubule protein upon addition of tubulin to short pieces of double-walled microtubules either their inner or their outer wall elongates resulting in normal microtubules or in macrotubules, respectively. Because of their genesis we suggest that macrotubules like double-walled microtubules (see Unger et al., Eur. J. Cell Biol. 46, 98-104 (1988)) expose those sides of tubulin dimers at their surface which usually form the lumen face of microtubules.     

Localization of the Trypanosoma cruzi-specific Mr 25,000 antigen by immune electron microscopy using monoclonal antibodies

Two monoclonal antibodies reacted with the Trypanosoma cruzi-specific antigen of an apparent Mr 25,000 from all developmental forms (Tachibana et al. 1986). This T. cruzi-specific antigen was found at the plasma membrane by immunoperoxidase electron microscopy using the monoclonal antibodies TCF48 and TCF87. The TCF48 and TCF87-treated cells showed stain deposits at the plasma membrane clearly distinguishable from those in cells treated with a monoclonal antibody against a surface antigen. This suggests that the epitope(s) of the Mr 25,000 antigen is located on the inner surface or in the matrix of the plasma membrane. TCF48 and TCF87 also reacted with an antigen on the microtubules of the axoneme, but not with the subpellicular microtubules. These results suggest that the T. cruzi-specific Mr 25,000 antigen is common to both the plasma membrane and axoneme but it is not located at the subpellicular microtubules. Its identity and that of the surface antigen, Gp25 (Scharfstein et al. 1983) as well as its role in the pathogenicity of the parasite are discussed.     

The centrosome opens the way to mitosis

During mitosis, the interaction between chromosomes and microtubules requires nuclear envelope disassembly in prophase. Two articles in this issue of Developmental Cell show that centrosomes have a role in promoting nuclear envelope breakdown (Hachet et al., 2007; Portier et al., 2007). Surprisingly, the role of the centrosome in this process is independent of its role as a microtubule nucleation organelle. Instead, the centrosome seems to act as a spatial regulator for the activation of the Aurora A kinase.     

Highways for mRNA transport

Two new studies reveal the role of microtubule polarity in the asymmetric localization of mRNAs. In this issue of Cell, Zimyanin et al. (2008) show that the asymmetric localization of oskar mRNA in fruit fly oocytes results from a slight bias in the direction of its transport. Meanwhile, Messitt et al. (2008) reporting in Developmental Cell find a subpopulation of microtubules that is critical for the asymmetric distribution of Vg1 mRNA in frog oocytes.     

Dynein at the nuclear envelope

Most cellular organelles are positioned through active transport by motor proteins. The authors discuss the evidence that dynein has important cell cycle-regulated functions in this context at the nuclear envelope.Most cellular organelles are positioned through active transport by motor proteins. This is especially important during cell division, a time when the organelles and genetic content need to be divided equally between the two daughter cells. Although individual proteins can attain their correct location by diffusion, larger structures are usually positioned through active transport by motor proteins. The main motor that transports cargoes to the minus ends of the microtubules is dynein. In nondividing cells, dynein probably transports or positions the nucleus inside the cells by binding to the nuclear envelope (NE; Burke & Roux, 2009). However, it appears that dynein also has important cell-cycle-regulated functions at the NE, as it is recruited to the NE every cell cycle just before cells enter mitosis (Salina et al, 2002; Splinter et al, 2010). Here, we discuss why dynein might be recruited to the NE for a brief period before mitosis.During late G2 or prophase the centrosomes separate to opposite sides of the nucleus, but remain closely associated with the NE during separation. This close association is probably mediated through NE-bound dynein, which ‘walks'' towards the minus ends of centrosomal microtubules, thereby pulling centrosomes towards the NE (Splinter et al, 2010; Gonczy et al, 1999; Robinson et al, 1999). We speculate that close association of centrosomes to the NE might have several functions. First, if centrosomes are not mechanically coupled to the NE, centrosome movement during separation will occur in random directions and chromosomes will not end up between the two separated centrosomes. In this scenario, individual kinetochores might attach more frequently to microtubules coming from both centrosomes (merotelic attachments), a defect that can result in aneuploidy, a characteristic of cancer. Second, centrosome-nuclear attachment also keeps centrosomes in close proximity to chromosomes, which might facilitate rapid capture of chromosomes by microtubules nucleated by the centrosomes after NE breakdown. This might not be absolutely essential, as chromosome alignment can occur in the absence of centrosomes. However, the spatial proximity of centrosomes and chromosomes at NE breakdown might improve the fidelity of kinetochore capture and chromosome alignment.In addition, dynein has also been suggested to promote centrosome separation in prophase in some systems (Gonczy et al, 1999; Robinson et al, 1999; Vaisberg et al, 1993), although not in others (Tanenbaum et al, 2008). Perhaps dynein, anchored at the NE just before mitosis, could exert force on microtubules emanating from both centrosomes, thereby pulling centrosomes apart. However, this force could also be produced by cortical dynein and specific inhibition of NE-associated or cortical dynein will be required to test which pool is responsible.Dynein has also been implicated in the process of NE breakdown itself, by promoting mechanical shearing of the NE. Two elegant studies showed that microtubules can tear the NE as cells enter mitosis (Salina et al, 2002; Beaudouin et al, 2002). One possibility is that microtubules growing into the NE mechanically disrupt it. Alternatively, NE-associated dynein might ‘walk'' along centrosomal microtubules and thereby pull on the NE, tearing it apart. However, testing the exact role of dynein in NE breakdown is complicated by the fact that centrosomes detach from the NE on inactivation of dynein and centrosomal microtubules stop growing efficiently into the NE. Thus, selective inhibition of dynein function will also be required to test this idea.Specific recruitment of dynein to the NE just before mitosis clearly suggests a role for dynein at the NE in preparing cells for mitosis. A major role of NE-associated dynein is to maintain close association of centrosomes with the NE during centrosome separation, which might be needed for efficient capture and alignment of chromosomes after NE breakdown, but additionally, NE-associated dynein could facilitate breakdown and contribute to centrosome separation in some systems.     

ANALYSIS OF MICROTUBULE STRUCTURE IN EUGLENA GRANULATA

The microtubules associated with the pellicle, reservoir and locomotory flagellum of Euglena granulata were subjected to rotational analysis using the technique of Markham et al. The results indicate that the microtubules have 11-14 subunits when viewed in transverse section. Rotational analysis of the entire locomotory flagellum was also conducted to better demonstrate the nature of the arms extending from the peripheral microtubules.     

Cytokinetic astralogy

Division plane specification in animal cells has long been presumed to involve direct contact between microtubules of the anaphase mitotic spindle and the cell cortex. In this issue, von Dassow et al. (von Dassow et al. 2009. J. Cell. Biol. doi:10.1083/jcb.200907090) challenge this assumption by showing that spindle microtubules can effectively position the division plane at a distance from the cell cortex.Cell division, or cytokinesis, is accomplished via constriction of an equatorially localized contractile ring composed of filamentous actin and myosin II (Rappaport, 1996). Accurate division plane specification is essential to properly partition the cytoplasm and permit each daughter cell to receive a single copy of the genome. To ensure this accuracy, microtubules of the mitotic spindle signal to the cell cortex upon anaphase onset and promote assembly of the contractile ring between the separating chromosomes. The precise mechanism by which microtubules position the contractile ring, however, remains elusive.Early models on the nature of the spindle-derived signal proposed that astral rays (later found to be microtubules) position the division plane by either locally promoting contractility at the cell equator or inhibiting contractility at the cell poles (Rappaport, 1996). Recent evidence, though, suggests that distinct microtubule populations within a single cell provide multiple signals to promote accurate division (Canman et al., 2003; Bringmann and Hyman, 2005; Chen et al., 2008; Foe and von Dassow, 2008; von Dassow, 2009).The anaphase mitotic spindle contains several subtypes of microtubules, each of which is likely to contribute to division plane specification. Although kinetochore microtubules drive chromosome segregation during anaphase, nonkinetochore microtubules extend and maintain close proximity with the assembling central spindle (Mastronarde et al., 1993). Central spindle microtubules are highly stable (Salmon et al., 1976) and organize into an antiparallel bundled array between the separating chromosomes (Mastronarde et al., 1993). Preventing central spindle assembly usually results in a complete failure in cytokinesis, and prevents division plane specification in many cell types (Glotzer, 2005). Astral microtubules, however, are highly dynamic and grow out circumferentially from the centrosomes toward the cell cortex. Increasing evidence suggests that the astral microtubule signal inhibits contractility (see below; Canman et al., 2000; Kurz et al., 2002; Lewellyn et al., 2009).Regardless of the mechanism of division plane specification via microtubules, nearly all current models depend on direct contact between microtubules of the mitotic spindle and the cell cortex. Most of these models were based on observations that at the time of division plane specification, astral microtubules contact the cell cortex in nearly all systems studied. Nonkinetochore and/or central spindle microtubules have also been proposed to deliver critical contractile signals to the cell equator (Murata-Hori and Wang, 2002; Canman et al., 2003; Somers and Saint, 2003; Verbrugghe and White, 2004; Lewellyn et al., 2009; Vale et al., 2009). Yet in many cell types (especially early embryos), central spindle microtubules are at some distance from the cell cortex during division plane specification. Despite this, signal delivery for both astral and central spindle microtubules was proposed to occur via direct transport along microtubules to the cell cortex. The study of von Dassow et al. in this issue, however, indicates that accurate division plane specification does not require any close microtubule/cortical contact and may occur via a diffusion-based mechanism (see also Salmon and Wolniak, 1990).By treating echinoderm and Xenopus embryos with controlled levels of trichostatin A (TSA), which destabilizes acetylated dynamic microtubules via inhibition of the tubulin deacetylase HDAC6 (Matsuyama et al., 2002), von Dassow et al. (2009) were able to preferentially prevent astral microtubule growth while leaving central spindle microtubules intact. TSA treatment did not block anaphase onset or central spindle assembly, but resulted in the complete disruption of all direct microtubule contact with the cell cortex. Nevertheless, TSA-treated cells were able to undergo cytokinesis successfully (Fig. 1 A). The lack of astral microtubules in TSA-treated cells was also recapitulated by double centrosome ablation, and again the cells were able to undergo cytokinesis (Fig. 1 B). In both experiments, cytokinesis occurred in a timely manner, but the contractile ring was broader than in control cells. Together, these data suggest that spindle microtubules are sufficient to provide a diffusible stimulatory signal capable of defining the cell division plane without any direct contact with the cell cortex (von Dassow et al., 2009).Open in a separate windowFigure 1.Testing models of division plane specification by targeting distinct microtubule populations. By selectively eliminating astral microtubules with either controlled TSA-treatment (A) or by double centrosome ablation (B), von Dassow et al. (2009) provide strong evidence that microtubule contact with the cell cortex is not essential for successful cytokinesis. When a single centrosome was ablated, the division plane was displaced away from the ablated aster (B); this suggests that astral microtubules provide an inhibitory signal. Further, anucleate cells would only complete cytokinesis if the intracentrosomal distance exceeded the distance from the centrosomes to the cell cortex (C).The authors noticed that cytokinesis occurred selectively at a position with reduced microtubule density in control cells; therefore, they explored the role of astral microtubules in division plane positioning. By selectively ablating one centrosome just before anaphase onset, von Dassow et al. (2009) were also able to provide strong support for an inhibitory role of astral microtubules in division plane specification. When a single centrosome was ablated, the division plane was displaced away from the remaining astral microtubules and toward the ablated centrosome (Fig. 1 B). Further evidence for an inhibitory role of astral microtubules in cytokinesis came from close examination of the intracentrosomal distance in anucleate cells that were able to undergo cytokinesis relative to those that were not. Cells were only able to undergo cytokinesis when the intracentrosomal distance exceeded the distance from the centrosomes to the cell cortex (Fig. 1 C), which suggests that cytokinesis requires an aster-free zone. The authors propose a mechanism in these anucleate cells whereby global activation of contractility drives division plane specification refined by a zone of astral separation (von Dassow et al., 2009). However, one possibility is that a central spindle still forms in these anucleate cells and thus provides the same diffusion-based signal that promotes division in cells without asters. Indeed, antiparallel arrays of bundled microtubules that resemble the central spindle are known to form between asters without intervening chromosomes in other systems (Savoian et al., 1999).To summarize, the results described by von Dassow et al. (2009) support a model in which central spindle microtubules provide a diffusible stimulatory signal to promote the assembly of a broad contractile ring, which is then refined by astral microtubules into a tight contractile ring. It is tempting to speculate on the molecular nature of this diffusible signal and mechanism of the astral refinement during cytokinesis. Signaling via the small GTPase Rho is required for cytokinesis and is dependent on spindle microtubules (Bement et al., 2005; Piekny et al., 2005). von Dassow et al. (2009) showed that in TSA-treated cells lacking astral microtubules, the equatorial zone of active Rho GTPase is broader relative to control cells. Rho activation is promoted (at least in part) via the central spindle–localized GTP exchange factor, ECT2 (Glotzer, 2005). In parallel, the GTPase-activating protein (GAP) CYK4/MgcRacGAP also associates with the central spindle, where it acts to both limit the zone of Rho activity (Miller and Bement, 2009) and to promote the inactivation of another small GTPase, Rac (D''Avino et al., 2004; Yoshizaki et al., 2004; Canman et al., 2008). Perhaps in parallel to central spindle mediated activation of Rho signaling, local inactivation of the inhibitory Rac signal via CYK4 GAP activity would further specify the division plane, even at a distance (Fig. 2). When the dynamic asters are present, they could then additionally amplify Rac signaling at the cell poles via a similar mechanism to what occurs during cell motility (Wittmann and Waterman-Storer, 2001). This local feedback loop would reinforce the positive signal coming from the central spindle via Rho activation and could help delimit active Rho at the cell equator (Fig. 2). Certainly, understanding how Rho activation can be propagated to the cell cortex via diffusion in such an accurate manner will be a major future challenge.Open in a separate windowFigure 2.Model for central spindle–mediated signaling via Rho family small GTPases. Central spindle–localized guanine nucleotide exchange factor ECT2 leads to Rho activation at the cell equator. At the same time, central spindle–localized CYK4 (a Rho family GAP) would also locally inactivate the inhibitory Rac signal. Further refinement of the zone of active Rho by astral microtubule–activated Rac could then sharpen the Rho zone into a tight contractile ring.     

Cortical Microtubule Arrays Are Initiated from a Nonrandom Prepattern Driven by Atypical Microtubule Initiation

The ordered arrangement of cortical microtubules in growing plant cells is essential for anisotropic cell expansion and, hence, for plant morphogenesis. These arrays are dismantled when the microtubule cytoskeleton is rearranged during mitosis and reassembled following completion of cytokinesis. The reassembly of the cortical array has often been considered as initiating from a state of randomness, from which order arises at least partly through self-organizing mechanisms. However, some studies have shown evidence for ordering at early stages of array assembly. To investigate how cortical arrays are initiated in higher plant cells, we performed live-cell imaging studies of cortical array assembly in tobacco (Nicotiana tabacum) Bright Yellow-2 cells after cytokinesis and drug-induced disassembly. We found that cortical arrays in both cases did not initiate randomly but with a significant overrepresentation of microtubules at diagonal angles with respect to the cell axis, which coincides with the predominant orientation of the microtubules before their disappearance from the cell cortex in preprophase. In Arabidopsis (Arabidopsis thaliana) root cells, recovery from drug-induced disassembly was also nonrandom and correlated with the organization of the previous array, although no diagonal bias was observed in these cells. Surprisingly, during initiation, only about one-half of the new microtubules were nucleated from locations marked by green fluorescent protein-γ-tubulin complex protein2-tagged γ-nucleation complexes (γ-tubulin ring complex), therefore indicating that a large proportion of early polymers was initiated by a noncanonical mechanism not involving γ-tubulin ring complex. Simulation studies indicate that the high rate of noncanonical initiation of new microtubules has the potential to accelerate the rate of array repopulation.Higher plant cells feature ordered arrays of microtubules at the cell cortex (Ledbetter and Porter, 1963) that are essential for cell and tissue morphogenesis, as revealed by disruption of cortical arrays by drugs that cause microtubule depolymerization (Green, 1962) or stabilization (Weerdenburg and Seagull, 1988) and by loss-of-function mutations in a wide variety of microtubule-associated proteins (Baskin, 2001; Whittington et al., 2001; Buschmann and Lloyd, 2008; Lucas et al., 2011). The structure of these arrays is thought to control the pattern of cell growth primarily by its role in the deposition of cellulose microfibrils, the load-bearing component of the cell wall (Somerville, 2006). Functional relations between cortical microtubules and cellulose microfibrils have been proposed since the early sixties, even before cortical microtubules had been visualized (Green, 1962). Recent live-cell imaging studies have confirmed that cortical microtubules indeed guide the movement of cellulose synthase complexes that produce cellulose microfibrils (Paredez et al., 2006) and have shown further that microtubules position the insertion of most cellulose synthase complexes into the plasma membrane (Gutierrez et al., 2009). These activities of ordered cortical microtubules are proposed to facilitate the organization of cell wall structure, creating material properties that underlie cell growth anisotropy.While organization of the interphase cortical array appears to be essential for cell morphogenesis, this organization is disrupted during the cell cycle as microtubules are rearranged to create the preprophase band, spindle, and phragmoplast during mitosis and cytokinesis (for review, see Wasteneys, 2002). Upon completion of cytokinesis, an organized interphase cortical array is regenerated, but the pathway for this reassembly is not well understood.The plant interphase microtubule array is organized and maintained without centrosomes as organizing centers (for review, see Wasteneys, 2002; Bartolini and Gundersen, 2006; Ehrhardt and Shaw, 2006), and microtubule self-organization is proposed to play an important role in cortical microtubule array ordering (Dixit and Cyr, 2004). In electron micrographs, microtubules have been observed to be closely associated with the plasma membrane (Hardham and Gunning, 1978), and live-cell imaging provides evidence for attachment of microtubules to the cell cortex (Shaw et al., 2003; Vos et al., 2004). The close association to the plasma membrane restricts the cortical microtubules to a quasi two-dimensional plane where they interact through polymerization-driven collisions (Shaw et al., 2003; Dixit and Cyr, 2004). Microtubule encounters at shallow angles (<40°) have a high probability of leading to bundling, while microtubule encounters at steeper angles most likely result in induced catastrophes or microtubule crossovers (Dixit and Cyr, 2004). Several computational modeling studies have since shown that these types of interactions between surface-bound dynamical microtubules can indeed explain spontaneous coalignment of microtubules (Allard et al., 2010; Eren et al., 2010; Hawkins et al., 2010; Tindemans et al., 2010).The question of how the orientation of the cortical array is established with respect to the cell axis is less well understood. One possibility is that microtubules are selectively destabilized with respect to cellular coordinates (Ehrhardt and Shaw, 2006). Indeed, recent results from biological observations and modeling suggest that catastrophic collisions induced at the edges between cell faces or heighted catastrophe rates in cell caps could be sufficient to selectively favor microtubules in certain orientation and hence determine the final orientation of the array (Allard et al., 2010; Eren et al., 2010; Ambrose et al., 2011; Dhonukshe et al., 2012).To date, all models of cortical array assembly assume random initial conditions. However, experimental work by Wasteneys and Williamson (1989a, 1989b) in Nitella tasmanica showed that, during array reassembly after drug-induced disruption, microtubules were initially transverse. This was followed by a less ordered phase and later by the acquisition of the final transverse order. A nonrandom initial ordering was also observed in tobacco (Nicotiana tabacum) Bright Yellow-2 (BY-2) cells by Kumagai et al. (2001), who concluded that the process of transverse array establishment starts with longitudinal order but did not provide quantitative data for the process of array assembly. The initial conditions for the cortical microtubule array formation are important to consider, as they may strongly influence the speed at which order is established and could even prevent it from being established over a biologically relevant time scale.In this study, we used live-cell imaging to follow and record the whole transition from the cortical microtubule-free state to the final transverse array and used digital tracking algorithms to quantify the microtubule order. Nucleation stands out as a central process to characterize during array initiation. Lacking a central body to organize microtubule nucleations, the higher plant cell has dispersed nucleation complexes (Wasteneys and Williamson, 1989a, 1989b; Chan et al., 2003; Shaw et al., 2003; Murata et al., 2005; Pastuglia et al., 2006; Nakamura et al., 2010). Therefore, we performed high time resolution observations to quantify nucleation complex recruitment, nucleation rates, and microtubule nucleation angles. We found evidence for a highly nonrandom initial ordering state that features diagonal microtubule orientation and an atypical microtubule initiation mechanism. Simulation analysis indicates that these atypical nucleations have the potential to accelerate the recovery of cortical array density.     

Binding of kinesin to stress fibers in fibroblasts under condition of microtubule depolymerization

The localization of kinesin in EBTr (bovine embryonic trachea fibroblast) cells was studied by indirect immunofluorescence microscopy using an affinity-purified antibody against bovine adrenal kinesin. It has already been shown that in interphase cells a part of kinesin is located on microtubules and the rest diffusely distributed throughout the cytoplasm [Murofushi et al., 1988]. When microtubules were depolymerized with cold or colchicine treatment, antikinesin antibody-stained fibrous components distinct from microtubules. These fibrous structures were considered to be stress fibers because they were stained with rhodamine-phalloidin and because the fibrous staining with antikinesin antibody was completely lost by treating the cells with cytochalasin D along with colchicine. When cold-treated cells in which a major part of kinesin had been localized on stress fibers were incubated at 37 degrees C, kinesin reappeared on reconstituted microtubules. These observations strongly suggest that kinesin has affinity not only to microtubules but also to stress fibers in culture cells.     

Vibration and length-dependent flexural rigidity of protein microtubules using higher order shear deformation theory

Microtubules are hollow cylindrical filaments of the eukaryotic cytoskeleton characterized by extremely low shear modulus. A remarkable controversy has occurred in the literature, regarding the length dependence of flexural rigidity of microtubules predicted by the classical elastic beam model. In this study, a higher order shear deformable beam model for microtubules is employed to study unexplained length-dependent flexural rigidity and Young’s modulus of microtubules reported in the literature. The formulation allows for warping of the cross-section of the microtubule and eliminates the need for using arbitrary shear correction coefficients as in other theories. It is showed that vibration frequencies predicted by the present parabolic shear deformation theory (PSDT) are much lower than that given by the approximate isotropic beam model for shorter microtubules, although the two models give almost identical results for sufficiently long microtubules. It is confirmed that transverse shearing and the warping of the cross-section of microtubules are mainly responsible for the length-dependent flexural rigidity of an isolated microtubule reported in the literature, which cannot be explained by the widely used Euler-Bernoulli beam model. Indeed, the length-dependent flexural rigidity predicted by the present model is found to be in qualitative agreement with the existing experimental data ( [Kurachi et al., 1995] and [Pampaloni et al., 2006]). These results recommend that the parabolic shear deformation-beam theory offers a unified simple 1D model, which can capture the length dependence of flexural rigidity and be applied to various static and dynamic problems of microtubule mechanics.     

Characterization of smooth muscle caldesmon as a microtubule-associated protein.

We have previously shown that nonmuscle caldesmon copurified with brain microtubules binds to microtubules in vitro [Ishikawa et al.: FEBS Lett. 299:54-56, 1992]. To explore the role of caldesmon in the functions of microtubules, further characterization was performed using smooth muscle caldesmon, whose molecular structure and function have been best-characterized in all caldesmon species. Smooth muscle caldesmon bound to microtubules with a stoichiometry of five tubulin dimers to one molecule of caldesmon with the binding constant of 1.1 x 10(6) M-1. The binding of caldesmon to microtubules was inhibited in the presence of Ca2+ and calmodulin. Partial digestion of the caldesmon with alpha-chymotrypsin revealed that the binding site of the caldesmon for microtubules lay in the 34-kDa C-terminal domain. When the caldesmon was in the dimeric form in the absence of a reducing agent, the caldesmon cross-linked microtubules to form bundles. Further, the caldesmon potentiated the polymerization of tubulin, and inhibited the in vitro movement of microtubules on dynein. These results suggest that caldesmon may be involved in the regulation by Ca2+ of the functions of microtubules.     

The Alzheimer-like phosphorylation of tau protein reduces microtubule binding and involves Ser-Pro and Thr-Pro motifs.

Tau protein can be transformed into an Alzheimer-like state by phosphorylation with a kinase activity from brain [Biernat et al. (1992) EMBO J. 11, 1593-1597]. Here we show that the phosphorylation at Ser-Pro motifs strongly decreases tau's affinity for microtubules. The major reduction occurs during the first of the three main stages of phosphorylation. The data explain the lower stability of microtubules resulting from the pathological tau phosphorylation.