Microtubules: Sizing Up the GTP Cap

SummaryThe ‘GTP cap’ of the microtubule has long been postulated to exist, but a recent experiment gives us the first quantitative measurements of the cap size in the cell

CLASP promotes microtubule rescue by recruiting tubulin dimers to the microtubule

Spatial regulation of microtubule (MT) dynamics contributes to cell polarity and cell division. MT rescue, in which a MT stops shrinking and reinitiates growth, is the least understood aspect of MT dynamics. Cytoplasmic Linker Associated Proteins (CLASPs) are a conserved class of MT-associated proteins that contribute to MT stabilization and rescue in vivo. We show here that the Schizosaccharomyces pombe CLASP, Cls1p, is a homodimer that binds an αβ-tubulin heterodimer through conserved TOG-like domains. In vitro, CLASP increases MT rescue frequency, decreases MT catastrophe frequency, and moderately decreases MT disassembly rate. CLASP binds stably to the MT lattice, recruits tubulin, and locally promotes rescues. Mutations in the CLASP TOG domains demonstrate that tubulin binding is critical for its rescue activity. We propose a mechanism for rescue in which CLASP-tubulin dimer complexes bind along the MT lattice and reverse MT depolymerization with their bound tubulin dimer

CLASP promotes microtubule rescue by recruiting tubulin dimers to the microtubule

Spatial regulation of microtubule (MT) dynamics contributes to cell polarity and cell division. MT rescue, in which a MT stops shrinking and reinitiates growth, is the least understood aspect of MT dynamics. Cytoplasmic Linker Associated Proteins (CLASPs) are a conserved class of MT-associated proteins that contribute to MT stabilization and rescue in vivo. We show here that the Schizosaccharomyces pombe CLASP, Cls1p, is a homodimer that binds an αβ-tubulin heterodimer through conserved TOG-like domains. In vitro, CLASP increases MT rescue frequency, decreases MT catastrophe frequency, and moderately decreases MT disassembly rate. CLASP binds stably to the MT lattice, recruits tubulin, and locally promotes rescues. Mutations in the CLASP TOG domains demonstrate that tubulin binding is critical for its rescue activity. We propose a mechanism for rescue in which CLASP-tubulin dimer complexes bind along the MT lattice and reverse MT depolymerization with their bound tubulin dimer

XMAP215 is a Processive Microtubule Polymerase

Fast growth of microtubules is essential for rapid assembly of the microtubule cytoskeleton during cell proliferation and differentiation. XMAP215 belongs to a conserved family of proteins that promote microtubule growth. To determine how XMAP215 accelerates growth, we developed a single-molecule assay to visualize directly XMAP215-GFP interacting with dynamic microtubules. XMAP215 binds free tubulin in a 1:1 complex that interacts with the microtubule lattice and targets the ends by a diffusion-facilitated mechanism. XMAP215 persists at the plus end for many rounds of tubulin subunit addition in a form of “tip-tracking.” These results show that XMAP215 is a processive polymerase that directly catalyzes the addition of up to 25 tubulin dimers to the growing plus end. Under some circumstances XMAP215 can also catalyze the reverse reaction, namely microtubule shrinkage. The similarities between XMAP215 and formins, actin polymerases, suggest that processive tip-tracking is a common mechanism for stimulating the growth of cytoskeletal polymers.Molecular and Cellular Biolog

Polar ejection forces in mitosis.

During mitosis, polar ejection forces (PEFs) are hypothesized to direct prometaphase chromosomes movements by pushing chromosome arms toward the spindle equator. PEFs are postulated to be caused by (a) plus-end directed microtubule (MT) based motor proteins on the chromosome arms, namely chromokinesins, and (b) the polymerization of spindle MTs into the chromosome. However, the exact role of PEFs is unclear, since little is known about the magnitude or form of PEFs. This study employs optical tweezers to recreate the lateral interaction between chromosome arms and MTs in vitro to obtain the first direct measurement of the speed and force of the PEFs developed on chromosome arms. The results include forces that frequently exceed 1 pN, maximum forces of 2--3 pN, and velocities of 83 +/- 56 nm/s; the movements exhibit a characteristic, non-continuous motion that includes displacements of >50 nm, stalls, and backwards slippage of the MT even under low loads. This activity is attributed to chromokinesin motors based on its ATP-dependence, antibody blocking experiments, and quantitative fluorescence. At first glance, this motor activity appears surprisingly weak and erratic, but it is ideally suited for producing PEFs that guide chromosome movements without severely deforming or damaging the local chromosome structure.Ph.D.Applied SciencesBiological SciencesBiomedical engineeringBiophysicsCellular biologyUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/124370/2/3138121.pd

Motors and MAPs Collaborate to Size Up Microtubules

Midzone microtubules keep chromosomes apart after segregation and provide a platform for cytokinesis factors. Reporting recently in Cell, Subramanian et al. (2013) describe how the motor protein kinesin-4 and the microtubule-associated protein PRC1 work together to mark microtubule ends for incorporation into the midzone in a length-dependent manner

The depolymerizing kinesin MCAK uses lattice diffusion to rapidly target microtubule ends

The microtubule cytoskeleton is a dynamic structure in which the lengths of the microtubules are tightly regulated. One regulatory mechanism is the depolymerization of microtubules by motor proteins in the kinesin-13 family1. These proteins are crucial for the control of microtubule length in cell division2–4, neuronal development5 and interphase microtubule dynamics6,7. The mechanism by which kinesin-13 proteins depolymerize microtubules is poorly understood. A central question is how these proteins target to microtubule ends at rates exceeding those of standard enzyme–substrate kinetics8. To address this question we developed a single-molecule microscopy assay for MCAK, the founding member of the kinesin-13 family9. Here we show that MCAK moves along the microtubule lattice in a one-dimensional (1D) random walk. MCAK–microtubule interactions were transient: the average MCAK molecule diffused for 0.83 s with

Loss of function of the Drosophila

The centrosome-associated proteins Ninein (Nin) and Ninein-like protein (Nlp) play significant roles in microtubule stability, nucleation and anchoring at the centrosome in mammalian cells. Here, we investigate Blastoderm specific gene 25D (Bsg25D), which encodes the only Drosophila protein that is closely related to Nin and Nlp. In early embryos, we find that Bsg25D mRNA and Bsg25D protein are closely associated with centrosomes and astral microtubules. We show that sequences within the coding region and 3′UTR of Bsg25D mRNAs are important for proper localization of this transcript in oogenesis and embryogenesis. Ectopic expression of eGFP-Bsg25D from an unlocalized mRNA disrupts microtubule polarity in mid-oogenesis and compromises the distribution of the axis polarity determinant Gurken. Using total internal reflection fluorescence microscopy, we show that an N-terminal fragment of Bsg25D can bind microtubules in vitro and can move along them, predominantly toward minus-ends. While flies homozygous for a Bsg25D null mutation are viable and fertile, 70% of embryos lacking maternal and zygotic Bsg25D do not hatch and exhibit chromosome segregation defects, as well as detachment of centrosomes from mitotic spindles. We conclude that Bsg25D is a centrosomal protein that, while dispensable for viability, nevertheless helps ensure the integrity of mitotic divisions in Drosophila