Yeast kinesin-8 depolymerizes microtubules in a length-dependent manner

The microtubule cytoskeleton and the mitotic spindle are highly dynamic structures, yet their sizes are remarkably constant, thus indicating that the growth and shrinkage of their constituent microtubules are finely balanced. This balance is achieved, in part, through kinesin-8 proteins (such as Kip3p in budding yeast and KLP67A in Drosophila) that destabilize microtubules. Here, we directly demonstrate that Kip3p destabilizes microtubules by depolymerizing them--accounting for the effects of kinesin-8 perturbations on microtubule and spindle length observed in fungi and metazoan cells. Furthermore, using single-molecule microscopy assays, we show that Kip3p has several properties that distinguish it from other depolymerizing kinesins, such as the kinesin-13 MCAK. First, Kip3p disassembles microtubules exclusively at the plus end and second, remarkably, Kip3p depolymerizes longer microtubules faster than shorter ones. These properties are consequences of Kip3p being a highly processive, plus-end-directed motor, both in vitro and in vivo. Length-dependent depolymerization provides a new mechanism for controlling the lengths of subcellular structures.     

Effects of random hydrolysis on biofilament length distributions in a shared subunit pool

The sizes of filamentous structures in a cell are often regulated for many physiological processes. A key question in cell biology is how such size control is achieved. Here, we theoretically study the length distributions of multiple filaments, growing by stochastic assembly and disassembly of subunits from a limiting subunit pool. Importantly, we consider a chemical switching of subunits (hydrolysis) prevalent in many biofilaments like microtubules (MTs). We show by simulations of different models that hydrolysis leads to a skewed unimodal length distribution for a single MT. In contrast, hydrolysis can lead to bimodal distributions of individual lengths for two MTs, where individual filaments toggle stochastically between bigger and smaller sizes. For more than two MTs, length distributions are also bimodal, although the bimodality becomes less prominent. We further show that this collective phenomenon is connected with the nonequilibrium nature of hydrolysis, and the bimodality disappears for reversible dynamics. Consistent with earlier theoretical studies, a homogeneous subunit pool, without hydrolysis, cannot control filament lengths. We thus elucidate the role of hydrolysis as a control mechanism on MT length diversity.     

Analysis of flagellar size control using a mutant of Chlamydomonas reinhardtii with a variable number of flagella

A mutant of Chlamydomonas reinhardtii with a variable number of flagella per cell has been used to investigate flagellar size control. The mutant and wild-type do not differ in cell size nor in flagellar length, yet the size of the intracellular pool of flagellar precursor protein can differ dramatically among individual mutant cells, with, for example, triflagellate cells having three times the pool of monoflagellate cells. Because cells of the same size, but with very different pool sizes, have flagella of identical length, it appears that the concentration of the unassembled flagellar precursor protein pool does not regulate flagellar length. The relation between cell size, pool size, and flagellar length has also been investigated for wild-type cells of different sizes and ploidies. Again, flagellar length appears to be maintained independent of pool size or concentration.     

The phosphorylation state of an aurora-like kinase marks the length of growing flagella in Chlamydomonas

Flagella and cilia are structurally polarized organelles whose lengths are precisely defined, and alterations in length are related to several human disorders. Intraflagellar transport (IFT) and protein signaling molecules are implicated in specifying flagellar and ciliary length, but evidence has been lacking for a flagellum and cilium length sensor that could participate in active length control or establishment of structural polarity. Previously, we showed that the phosphorylation state of the aurora-like protein kinase CALK in Chlamydomonas is a marker of the absence of flagella. Here we show that CALK phosphorylation state is also a marker for flagellar length. CALK is phosphorylated in cells without flagella, and during flagellar assembly it becomes dephosphorylated. Dephosphorylation is not simply a consequence of initiation of flagellar assembly or of time after experimentally induced flagellar loss, but instead requires flagella to be assembled to a threshold length. Analysis of cells with flagella of varying lengths shows that the threshold length for CALK dephosphorylation is ~6 μm (half length). Studies with short and long flagellar mutants indicate that cells detect absolute rather than relative flagellar length. Our results demonstrate that cells possess a mechanism for translating flagellar length into a posttranslational modification of a known flagellar regulatory protein.     

Microtubule polymerases and depolymerases

The variety of shapes and sizes of the microtubule cytoskeleton is as great as the number of different cell types. This large variety is a consequence of the dynamic properties of microtubules, which allow them to adopt distributions of arbitrary size and form. How is the distribution of microtubule lengths controlled? Recent work suggests that the length distribution is controlled, at least in part, by the activity of microtubule polymerases and depolymerases, which accelerate microtubule growth and shrinkage. Specifically, biochemical and single-molecule studies have shown how MCAK (kinesin-13) and Kip3p (kinesin-8) accelerate depolymerization and how XMAP215 may accelerate growth. Studies on the yeast Dam1 complex have shown how proteins can couple a cellular structure, the kinetochore, to the ends of polymerizing and depolymerizing microtubules.     

Xenopus Actin Depolymerizing Factor/Cofilin (XAC) Is Responsible for the Turnover of Actin Filaments in Listeria monocytogenes Tails

In contrast to the slow rate of depolymerization of pure actin in vitro, populations of actin filaments in vivo turn over rapidly. Therefore, the rate of actin depolymerization must be accelerated by one or more factors in the cell. Since the actin dynamics in Listeria monocytogenes tails bear many similarities to those in the lamellipodia of moving cells, we have used Listeria as a model system to isolate factors required for regulating the rapid actin filament turnover involved in cell migration. Using a cell-free Xenopus egg extract system to reproduce the Listeria movement seen in a cell, we depleted candidate depolymerizing proteins and analyzed the effect that their removal had on the morphology of Listeria tails. Immunodepletion of Xenopus actin depolymerizing factor (ADF)/cofilin (XAC) from Xenopus egg extracts resulted in Listeria tails that were approximately five times longer than the tails from undepleted extracts. Depletion of XAC did not affect the tail assembly rate, suggesting that the increased tail length was caused by an inhibition of actin filament depolymerization. Immunodepletion of Xenopus gelsolin had no effect on either tail length or assembly rate. Addition of recombinant wild-type XAC or chick ADF protein to XAC-depleted extracts restored the tail length to that of control extracts, while addition of mutant ADF S3E that mimics the phosphorylated, inactive form of ADF did not reduce the tail length. Addition of excess wild-type XAC to Xenopus egg extracts reduced the length of Listeria tails to a limited extent. These observations show that XAC but not gelsolin is essential for depolymerizing actin filaments that rapidly turn over in Xenopus extracts. We also show that while the depolymerizing activities of XAC and Xenopus extract are effective at depolymerizing normal filaments containing ADP, they are unable to completely depolymerize actin filaments containing AMPPNP, a slowly hydrolyzible ATP analog. This observation suggests that the substrate for XAC is the ADP-bound subunit of actin and that the lifetime of a filament is controlled by its nucleotide content.     

KLP10A and KLP59C: The Dynamic Duo of Microtubule Depolymerization

Kinesin-13s are important effectors of microtubule depolymerization in cells. In a recent series of studies, we examined the roles played by kinesin-13s throughout the cell cycle in Drosophila. Our findings have revealed remarkable coordination between two family members, KLP10A and KLP59C, in which alterations in the relative targeting of these proteins allows them to participate in markedly different tasks at distinct points in the cell cycle. During mitosis, KLP10A and KLP59C function in parallel by targeting to and depolymerizing the opposite ends of kinetochore-associated microtubules, thereby driving poleward chromatid motility by a Pacman-Flux mechanism. Alternatively, during interphase, both proteins target to the same end of the microtubule but act in series to divide the labor of microtubule depolymerization. KLP10A initiates depolymerization while KLP59C perpetuates depolymerization after its initiation. Below, we detail these findings and examine some of their implications.     

A synthetic ancestral kinesin-13 depolymerizes microtubules faster than any natural depolymerizing kinesin

The activity of a kinesin is largely determined by the approximately 350 residue motor domain, and this region alone is sufficient to classify a kinesin as a member of a particular family. The kinesin-13 family are a group of microtubule depolymerizing kinesins and are vital regulators of microtubule length. Kinesin-13s are critical to spindle assembly and chromosome segregation in both mitotic and meiotic cell division and play crucial roles in cilium length control and neuronal development. To better understand the evolution of microtubule depolymerization activity, we created a synthetic ancestral kinesin-13 motor domain. This phylogenetically inferred ancestral motor domain is the sequence predicted to have existed in the common ancestor of the kinesin-13 family. Here we show that the ancestral kinesin-13 motor depolymerizes stabilized microtubules faster than any previously tested depolymerase. This potent activity is more than an order of magnitude faster than the most highly studied kinesin-13, MCAK and allows the ancestral kinesin-13 to depolymerize doubly stabilized microtubules and cause internal breaks within microtubules. These data suggest that the ancestor of the kinesin-13 family was a ‘super depolymerizer’ and that members of the kinesin-13 family have evolved away from this extreme depolymerizing activity to provide more controlled microtubule depolymerization activity in extant cells.     

Slow down of actin depolymerization by cross-linking molecules

The ability to control the assembly and disassembly dynamics of actin filaments is an essential property of the cellular cytoskeleton. While many different proteins are known which accelerate the polymerization of monomers into filaments or promote their disintegration, much less is known on mechanisms which guarantee the kinetic stability of the cytoskeletal filaments. Previous studies indicate that cross-linking molecules might fulfill these stabilizing tasks, which in addition facilitates their ability to regulate the organization of cytoskeletal structures in vivo. The effect of depolymerization factors on such structures or the mechanism which leads finally to their disintegration remain unknown. Here, we use multiple depolymerization methods in order to directly demonstrate that cross-linking and bundling proteins effectively suppress the actin depolymerization in a concentration dependent manner. Even the actin depolymerizing factor cofilin is not sufficient to facilitate a fast disintegration of highly cross-linked actin networks unless molecular motors are used simultaneously. The drastic modification of actin kinetics by cross-linking molecules can be expected to have wide-ranging implications for our understanding of the cytoskeleton, where cross-linking molecules are omnipresent and essential.     

Pivotal role of actin depolymerization in the regulation of cochlear outer hair cell motility

Cochlear outer hair cells undergo reversible changes in shape when externally stimulated. This response, known as OHC motility, is a central component of the cochlear amplifier, the mechanism responsible for the high sensitivity of mammalian hearing. We report that actin depolymerization, as regulated by activation/inhibition of LIMK/cofilin-mediated pathways, has a pivotal role in OHC motility. LIMK-mediated cofilin phosphorylation, which inhibits the actin depolymerizing activity of this protein, increases both electromotile amplitude and total length of guinea pig OHCs. In contrast, a decrease in cofilin phosphorylation reduces both OHC electromotile amplitude and OHC length. Experiments with acetylcholine and lysophosphatidic acid indicate that the effects of these agents on OHC motility are associated with regulation of cofilin phosphorylation via different signaling cascades. On the other hand, nonlinear capacitance measurements confirmed that all observed changes in OHC motile response were independent of the performance of the motor protein prestin. Altogether, these results strongly support the hypothesis that the cytoskeleton has a major role in the regulation of OHC motility, and identify actin depolymerization as a key process for modulating cochlear amplification.     

Tropomyosin prevents depolymerization of actin filaments from the pointed end

Regulation of the pointed, or slow-growing, end of actin filaments is essential to the regulation of filament length. The purpose of this study is to investigate the role of skeletal muscle tropomyosin (TM) in regulating pointed end assembly and disassembly in vitro. The effects of TM upon assembly and disassembly of actin monomers from the pointed filament end were measured using pyrenyl-actin fluorescence assays in which the barbed ends were capped by villin. Tropomyosin did not affect pointed end elongation; however, filament disassembly from the pointed end stopped in the presence of TM under conditions where control filaments disassembled within minutes. The degree of protection against depolymerization was dependent upon free TM concentration and upon filament length. When filaments were diluted to a subcritical actin concentration in TM, up to 95% of the filamentous actin remained after 24 h and did not depolymerize further. Longer actin filaments (150 monomers average length) were more effectively protected from depolymerization than short filaments (50 monomers average length). Although filaments stopped depolymerizing in the presence of TM, they were not capped as shown by elongation assays. This study demonstrates that a protein, such as TM, which binds to the side of the actin filament can prevent dissociation of monomers from the end without capping the end to elongation. In skeletal muscle, tropomyosin could prevent thin filament disassembly from the pointed end and constitute a mechanism for regulating filament length.     

Human Fidgetin is a microtubule severing the enzyme and minus-end depolymerase that regulates mitosis

Fidgetin is a member of the AAA protein superfamily with important roles in mammalian development. Here we show that human Fidgetin is a potent microtubule severing and depolymerizing the enzyme used to regulate mitotic spindle architecture, dynamics and anaphase A. In vitro, recombinant human Fidgetin severs taxol-stabilized microtubules along their length and promotes depolymerization, primarily from their minus-ends. In cells, human Fidgetin targets to centrosomes, and its depletion with siRNA significantly reduces the velocity of poleward tubulin flux and anaphase A chromatid-to-pole motion. In addition, the loss of Fidgetin induces a microtubule-dependent enlargement of mitotic centrosomes and an increase in the number and length of astral microtubules. Based on these data, we propose that human Fidgetin actively suppresses microtubule growth from and attachment to centrosomes.     

Human Fidgetin is a microtubule severing the enzyme and minus-end depolymerase that regulates mitosis

Fidgetin is a member of the AAA protein superfamily with important roles in mammalian development. Here we show that human Fidgetin is a potent microtubule severing and depolymerizing the enzyme used to regulate mitotic spindle architecture, dynamics and anaphase A. In vitro, recombinant human Fidgetin severs taxol-stabilized microtubules along their length and promotes depolymerization, primarily from their minus-ends. In cells, human Fidgetin targets to centrosomes, and its depletion with siRNA significantly reduces the velocity of poleward tubulin flux and anaphase A chromatid-to-pole motion. In addition, the loss of Fidgetin induces a microtubule-dependent enlargement of mitotic centrosomes and an increase in the number and length of astral microtubules. Based on these data, we propose that human Fidgetin actively suppresses microtubule growth from and attachment to centrosomes.     

Identification of XMAP215 as a microtubule-destabilizing factor in Xenopus egg extract by biochemical purification

Microtubules (MTs) polymerized with GMPCPP, a slowly hydrolyzable GTP analogue, are stable in buffer but are rapidly depolymerized in Xenopus egg extracts. This depolymerization is independent of three previously identified MT destabilizers (Op18, katanin, and XKCM1/KinI). We purified the factor responsible for this novel depolymerizing activity using biochemical fractionation and a visual activity assay and identified it as XMAP215, previously identified as a prominent MT growth-promoting protein in Xenopus extracts. Consistent with the purification results, we find that XMAP215 is necessary for GMPCPP-MT destabilization in extracts and that recombinant full-length XMAP215 as well as an NH2-terminal fragment have depolymerizing activity in vitro. Stimulation of depolymerization is specific for the MT plus end. These results provide evidence for a robust MT-destabilizing activity intrinsic to this microtubule-associated protein and suggest that destabilization may be part of its essential biochemical functions. We propose that the substrate in our assay, GMPCPP-stabilized MTs, serves as a model for the pause state of MT ends and that the multiple activities of XMAP215 are unified by a mechanism of antagonizing MT pauses.     

Functionally distinct kinesin-13 family members cooperate to regulate microtubule dynamics during interphase

Regulation of microtubule polymerization and depolymerization is required for proper cell development. Here, we report that two proteins of the Drosophila melanogaster kinesin-13 family, KLP10A and KLP59C, cooperate to drive microtubule depolymerization in interphase cells. Analyses of microtubule dynamics in S2 cells depleted of these proteins indicate that both proteins stimulate depolymerization, but alter distinct parameters of dynamic instability; KLP10A stimulates catastrophe (a switch from growth to shrinkage) whereas KLP59C suppresses rescue (a switch from shrinkage to growth). Moreover, immunofluorescence and live analyses of cells expressing tagged kinesins reveal that KLP10A and KLP59C target to polymerizing and depolymerizing microtubule plus ends, respectively. Our data also suggest that KLP10A is deposited on microtubules by the plus-end tracking protein, EB1. Our findings support a model in which these two members of the kinesin-13 family divide the labour of microtubule depolymerization.     

XKCM1 acts on a single protofilament and requires the C terminus of tubulin

The stability of microtubules during the cell-cycle is regulated by a number of cellular factors, some of which stabilize microtubules and others that promote breakdown. XKCM1 is a kinesin-like protein that induces microtubule depolymerization and is required for mitotic spindle assembly. We have examined the binding and depolymerization effects of XKCM1 on different tubulin polymers in order to learn about its mechanism of action. Zinc-induced tubulin polymers, characterized by an anti-parallel protofilament arrangement, are depolymerized by XKCM1, indicating that this enzyme acts on a single protofilament. GDP-tubulin rings, which correspond to the low-energy state of tubulin, are stable only under conditions that inhibit XKCM1 depolymerizing activity, but can be stabilized by XKCM1 bound to AMPPNP. Tubulin polymers made of subtilisin-treated tubulin (lacking the tubulin C-terminal tail) are resistant to XKCM1-induced depolymerization, suggesting that the interaction of the acidic tail of tubulin with basic residues in XKCM1 unique to Kin I proteins is required for depolymerization.     

Kinetic analysis of F-actin depolymerization in polymorphonuclear leukocyte lysates indicates that chemoattractant stimulation increases actin filament number without altering the filament length distribution

The rate of filamentous actin (F-actin) depolymerization is proportional to the number of filaments depolarizing and changes in the rate are proportional to changes in filament number. To determine the number and length of actin filaments in polymorphonuclear leukocytes and the change in filament number and length that occurs during the increase in F-actin upon chemoattractant stimulation, the time course of cellular F-actin depolymerization in lysates of control and peptide-stimulated cells was examined. F-actin was quantified by the TRITC-labeled phalloidin staining of pelletable actin. Lysis in 1.2 M KCl and 10 microM DNase I minimized the effects of F-actin binding proteins and G-actin, respectively, on the kinetics of depolymerization. To determine filament number and length from a depolymerization time course, depolymerization kinetics must be limited by the actin monomer dissociation rate. Comparison of time courses of depolymerization in the presence (pointed ends free) or absence (barbed and pointed ends free) of cytochalasin suggested depolymerization occurred from both ends of the filament and that monomer dissociation was rate limiting. Control cells had 1.7 +/- 0.4 x 10(5) filaments with an average length of 0.29 +/- 0.09 microns. Chemo-attractant stimulation for 90 s at room temperature with 0.02 microM N-formylnorleucylleucylphenylalanine caused a twofold increase in F-actin and about a two-fold increase in the total number of actin filaments to 4.0 +/- 0.5 x 10(5) filaments with an average length of 0.27 +/- 0.07 microns. In both cases, most (approximately 80%) of the filaments were quite short (less than or equal to 0.18 micron). The length distributions of actin filaments in stimulated and control cells were similar.     

Structure of a PL17 Family Alginate Lyase Demonstrates Functional Similarities among Exotype Depolymerases

Brown macroalgae represent an ideal source for complex polysaccharides that can be utilized as precursors for cellulosic biofuels. The lack of recalcitrant lignin components in macroalgae polysaccharide reserves provides a facile route for depolymerization of constituent polysaccharides into simple monosaccharides. The most abundant sugars in macroalgae are alginate, mannitol, and glucan, and although several classes of enzymes that can catabolize the latter two have been characterized, studies of alginate-depolymerizing enzymes have lagged. Here, we present several crystal structures of Alg17c from marine bacterium Saccharophagus degradans along with structure-function characterization of active site residues that are suggested to be involved in the exolytic mechanism of alginate depolymerization. This represents the first structural and biochemical characterization of a family 17 polysaccharide lyase enzyme. Despite the lack of appreciable sequence conservation, the structure and β-elimination mechanism for glycolytic bond cleavage by Alg17c are similar to those observed for family 15 polysaccharide lyases and other lyases. This work illuminates the evolutionary relationships among enzymes within this unexplored class of polysaccharide lyases and reinforces the notion of a structure-based hierarchy in the classification of these enzymes.     

Kinetochores use a novel mechanism for coordinating the dynamics of individual microtubules

Chromosome alignment during mitosis is frequently accompanied by a dynamic switching between elongation and shortening of kinetochore fibers (K-fibers) that connect kinetochores and spindle poles . In higher eukaryotes, mature K-fibers consist of 10-30 kinetochore microtubules (kMTs) whose plus ends are embedded in the kinetochore . A critical and long-standing question is how the dynamics of individual kMTs within the K-fiber are coordinated . We have addressed this question by using electron tomography to determine the polymerization/depolymerization status of individual kMTs in the K-fibers of PtK1 and Drosophila S2 cells. Surprisingly, we find that the plus ends of two-thirds of kMTs are in a depolymerizing state, even when the K-fiber exhibits net tubulin incorporation at the plus end . Furthermore, almost all individual K-fibers examined had a mixture of kMTs in the polymerizing and depolymerizing states. Therefore, although K-fibers elongate and shrink as a unit, the dynamics of individual kMTs within a K-fiber are not coordinated at any given moment. Our results suggest a novel control mechanism through which attachment to the kinetochore outer plate prevents shrinkage of kMTs. We discuss the ramifications of this new model on the regulation of chromosome movement and the stability of K-fibers.