Microtubule Sorting Research Articles

Multiple motors cooperate to establish and maintain acentrosomal spindle bipolarity in C. elegans oocyte meiosis.

While centrosomes organize spindle poles during mitosis, oocyte meiosis can occur in their absence. Spindles in human oocytes frequently fail to maintain bipolarity and consequently undergo chromosome segregation errors, making it important to understand the mechanisms that promote acentrosomal spindle stability. To this end, we have optimized the auxin-inducible degron system in Caenorhabditis elegans to remove the factors from pre-formed oocyte spindles within minutes and assess the effects on spindle structure. This approach revealed that dynein is required to maintain the integrity of acentrosomal poles; removal of dynein from bipolar spindles caused pole splaying, and when coupled with a monopolar spindle induced by depletion of the kinesin-12 motor KLP-18, dynein depletion led to a complete dissolution of the monopole. Surprisingly, we went on to discover that following monopole disruption, individual chromosomes were able to reorganize local microtubules and re-establish a miniature bipolar spindle that mediated chromosome segregation. This revealed the existence of redundant microtubule sorting forces that are undetectable when KLP-18 and dynein are active. We found that the kinesin-5 family motor BMK-1 provides this force, uncovering the first evidence that kinesin-5 contributes to C. elegans meiotic spindle organization. Altogether, our studies have revealed how multiple motors are working synchronously to establish and maintain bipolarity in the absence of centrosomes.

Kinetochore protein Spindly controls microtubule polarity in Drosophila axons

Microtubule polarity in axons and dendrites defines the direction of intracellular transport in neurons. Axons contain arrays of uniformly polarized microtubules with plus-ends facing the tips of the processes (plus-end-out), while dendrites contain microtubules with a minus-end-out orientation. It has been shown that cytoplasmic dynein, targeted to cortical actin, removes minus-end-out microtubules from axons. Here we have identified Spindly, a protein known for recruitment of dynein to kinetochores in mitosis, as a key factor required for dynein-dependent microtubule sorting in axons of Drosophila neurons. Depletion of Spindly affects polarity of axonal microtubules in vivo and in primary neuronal cultures. In addition to these defects, depletion of Spindly in neurons causes major collapse of axonal patterning in the third-instar larval brain as well as severe coordination impairment in adult flies. These defects can be fully rescued by full-length Spindly, but not by variants with mutations in its dynein-binding site. Biochemical analysis demonstrated that Spindly binds F-actin, suggesting that Spindly serves as a link between dynein and cortical actin in axons. Therefore, Spindly plays a critical role during neurodevelopment by mediating dynein-driven sorting of axonal microtubules.

Microtubule Sorting by Persistence Length and Surface Charge Density of Microtubules

Microtubule Sorting by Persistence Length and Surface Charge Density of Microtubules

Assembly of Caenorhabditis elegans acentrosomal spindles occurs without evident microtubule-organizing centers and requires microtubule sorting by KLP-18/kinesin-12 and MESP-1.

Although centrosomes contribute to spindle formation in most cell types, oocytes of many species are acentrosomal and must organize spindles in their absence. Here we investigate this process in Caenorhabditis elegans, detailing how acentrosomal spindles form and revealing mechanisms required to establish bipolarity. Using high-resolution imaging, we find that in meiosis I, microtubules initially form a "cage-like" structure inside the disassembling nuclear envelope. This structure reorganizes so that minus ends are sorted to the periphery of the array, forming multiple nascent poles that then coalesce until bipolarity is achieved. In meiosis II, microtubules nucleate in the vicinity of chromosomes but then undergo similar sorting and pole formation events. We further show that KLP-18/kinesin-12 and MESP-1, previously shown to be required for spindle bipolarity, likely contribute to bipolarity by sorting microtubules. After their depletion, minus ends are not sorted outward at the early stages of spindle assembly and instead converge. These proteins colocalize on microtubules, are interdependent for localization, and can interact, suggesting that they work together. We propose that KLP-18/kinesin-12 and MESP-1 form a complex that functions to sort microtubules of mixed polarity into a configuration in which minus ends are away from the chromosomes, enabling formation of nascent poles.

Maps and Motors Cooperate to form the Paraxial Microtubule Cytoskeleton in Differentiating Muscle Cells

The microtubule cytoskeleton is critical for muscle cell differentiation and undergoes reorganisation into an array of paraxial microtubules, which serve as templates for contractile sarcomere formation. The organisation of microtubules into a highly ordered array requires the cooperation of molecular motors and microtubule-associated proteins (MAPs). Undifferentiated muscle cells have a radial, centrosome-focussed microtubule array that is highly motile. The movement of microtubules is driven by kinesin-1 and dynein. Upon differentiation, microtubules become increasingly more stable, paraxially aligned and less motile. As kinesin-1 and dynein are active throughout differentiation, we hypothesised that a crosslinking MAP controls microtubule organisation and movement in differentiating muscle cells. We identified a previously uncharacterised isoform of microtubule-associated protein 4, oMAP4, as a microtubule organising factor crucial for myogenesis. We show that oMAP4 is expressed upon muscle cell differentiation and is the only MAP4 isoform essential for the morphological changes associated with muscle differentiation. Most notably, oMAP4 is required for paraxial microtubule organisation in muscle cells and prevents dynein- and kinesin-driven microtubule-microtubule sliding. Purified oMAP4 aligns dynamic microtubules into antiparallel bundles that withstand motor forces in vitro. Our data support a model in which the cooperation of dynein-mediated microtubule transport and oMAP4-mediated zippering of microtubules drives formation of a highly ordered paraxial microtubule array that provides critical support for the polarisation and elongation of myotubes. We believe this microtubule sorting mechanism is guided by the selective stabilisation of those microtubules that contact cell tips. Regulation of microtubule dynamics at the tips of elongating muscle cells is mediated by the MAP EB3 and results in the capture of microtubules so that these undergo rapid transitions between assembly and disassembly in the vicinity of the cell cortex for an extended period of time.

Nucleation and Transport Organize Microtubules in Metaphase Spindles

Spindles are arrays of microtubules that segregate chromosomes during cell division. It has been difficult to validate models of spindle assembly due to a lack of information on the organization of microtubules in these structures. Here we present a method, based on femtosecond laser ablation, capable of measuring the detailed architecture of spindles. We used this method to study the metaphase spindle in Xenopus laevis egg extracts and found that microtubules are shortest near poles and become progressively longer toward the center of the spindle. These data, in combination with mathematical modeling, imaging, and biochemical perturbations, are sufficient to reject previously proposed mechanisms of spindle assembly. Our results support a model of spindle assembly in which microtubule polymerization dynamics are not spatially regulated, and the proper organization of microtubules in the spindle is determined by nonuniform microtubule nucleation and the local sorting of microtubules by transport.

Microtubule organization by the antagonistic mitotic motors kinesin-5 and kinesin-14

During cell division, different molecular motors act synergistically to rearrange microtubules. Minus end-directed motors are thought to have a dual role: focusing microtubule ends to poles and establishing together with plus end-directed motors a balance of force between antiparallel microtubules in the spindle. We study here the competing action of Xenopus laevis kinesin-14 and -5 in vitro in situations in which these motors with opposite directionality cross-link and slide microtubules. We find that full-length kinesin-14 can form microtubule asters without additional factors, whereas kinesin-5 does not, likely reflecting an adaptation to mitotic function. A stable balance of force is not established between two antiparallel microtubules with these motors. Instead, directional instability is generated, promoting efficient motor and microtubule sorting. A nonmotor microtubule cross-linker can suppress directional instability but also impedes microtubule sorting, illustrating a conflict between stability and dynamicity of organization. These results establish the basic organizational properties of these antagonistic mitotic motors and a microtubule bundler.

Size sorting of kinesin-driven microtubules with topographical grooves on a chip

Gliding microtubules (MTs) on a surface coated with kinesin biomolecular motors have been suggested for the development of nanoscale transport systems. In order to establish a sorting function for gliding MTs, events for MTs approaching micro-scale grooves were investigated. MTs longer than the width of grooves fabricated on a Si substrate bridged the grooves (bridging) and many MTs shorter than the groove width almost began to bridge, but returned to the surface that they approached from (guiding). Occurrence probabilities for the events were analyzed with focus on the geometric conditions, such as length of the MTs, width of the grooves, and the incident angle (alpha) of the MTs approaching the grooves. The occurrence probability for bridging increased with an increase in the incident angle (16%, alpha = 0-30 degrees; 51%, alpha = 30-60 degrees; 75%, alpha = 60-90 degrees), and the probability for guiding decreased with an increase in the incident angle (79%, alpha = 0-30 degrees; 55%, alpha = 30-60 degrees; 5%, alpha = 60-90 degrees). The results indicate that an incident angle of 30-60 degrees is an effective condition for MT sorting, because the bridging and guiding events can sort MTs that are longer and shorter than the groove widths, respectively. Furthermore, the occurrence probabilities of both bridging and guiding in a higher concentration of methylcellulose (0.5%) increased up to approximately 70% at incident angles of 30-60 degrees, indicating good feasibility for the development of devices for the sorting of MTs on surfaces with topographical grooves.

The kinesin-14 Klp2 organizes microtubules into parallel bundles by an ATP-dependent sorting mechanism.

The dynamic organization of microtubules into parallel arrays allows interphase cells to set up multi-lane highways for intracellular transport and M-phase cells to build the mitotic and meiotic spindles. Here we show that a minimally reconstituted system composed of Klp2, a kinesin-14 from the fission yeast Schizosaccharomyces pombe, together with microtubules assembled from purified S. pombe tubulin, autonomously assembles bundles of parallel microtubules. Bundles form by an ATP-dependent sorting mechanism that requires the full-length Klp2 motor. By this mechanism, antiparallel-overlapped microtubules slide over one another until they dissociate from the bundles, whereas parallel-overlapped microtubules are selectively trapped by an energy-dissipating force-balance mechanism. Klp2-driven microtubule sorting provides a robust pathway for the organization of microtubules into parallel arrays. In vivo evidence indicates that Klp2 is required for the proper organization of S. pombe interphase microtubules into bipolar arrays of parallel-overlapped microtubules, suggesting that kinesin-14-dependent microtubule sorting may have wide biological importance.

326 分子モーターを用いた微小管長さソーティングに関する基礎検討(OS9-3:マイクロ・ナノバイオメカニクス(3),OS9:マイクロ・ナノバイオメカニクス)

326 分子モーターを用いた微小管長さソーティングに関する基礎検討(OS9-3:マイクロ・ナノバイオメカニクス(3),OS9:マイクロ・ナノバイオメカニクス)

Self-organization of microtubules into bipolar spindles around artificial chromosomes in Xenopus egg extracts.

Functional nuclei and mitotic spindles are shown to assemble around DNA-coated beads incubated in Xenopus egg extracts. Bipolar spindles assemble in the absence of centrosomes and kinetochores, indicating that bipolarity is an intrinsic property of microtubules assembling around chromatin in a mitotic cytoplasm. Microtubules nucleated at dispersed sites with random polarity rearrange into two arrays of uniform polarity. Spindle-pole formation requires cytoplasmic dynein-dependent translocation of microtubules across one another. It is proposed that spindles form in the absence of centrosomes by motor-dependent sorting of microtubules according to their polarity.