How germ granules promote germ cell fate.

Germ cells are the only cells in the body capable of giving rise to a new organism, and this totipotency hinges on their ability to assemble membraneless germ granules. These specialized RNA and protein complexes are hallmarks of germ cells throughout their life cycle: as embryonic germ granules in late oocytes and zygotes, Balbiani bodies in immature oocytes, and nuage in maturing gametes. Decades of developmental, genetic and biochemical studies have identified protein and RNA constituents unique to germ granules and have implicated these in germ cell identity, genome integrity and gamete differentiation. Now, emerging research is defining germ granules as biomolecular condensates that achieve high molecular concentrations by phase separation, and it is assigning distinct roles to germ granules during different stages of germline development. This organization of the germ cell cytoplasm into cellular subcompartments seems to be critical not only for the flawless continuity through the germline life cycle within the developing organism but also for the success of the next generation.

Coupling of microtubule bundles isolates them from local disruptions to set the structural stability of the anaphase spindle.

Chromosome segregation requires load-bearing interactions across kinetochore fibers and antiparallel microtubule bundles, which constitute the spindle midzone. Mechanical properties of kinetochore fibers have been characterized during metaphase, when the mitotic spindle achieves steady state. However, it has been difficult to probe the mechanics of the spindle midzone that elongates during anaphase. Here, we combine superresolution expansion and electron microscopies, lattice light-sheet imaging, and laser microsurgery to examine how midzone organization sets its mechanics. We find that individual midzone bundles extend out to multiple positions across chromosomes and form multiple apparent microtubule-based connections with each other. Across the spindle's short axis, these microtubule bundles exhibit restricted, submicrometer-amplitude motions, which are weakly correlated on <10s timescales. Severing individual midzone bundles near their center does not substantially affect positions of neighboring bundles, nor the overall structural stability of the midzone. In contrast, severing multiple midzone bundles or individual bundles at their chromosome-proximal ends significantly displaces neighboring microtubule bundles. Together, these data suggest a model wherein multiple midzone connections both reinforce its structure and mechanically isolate individual bundles from local perturbations. This feature sets the robust midzone architecture to accommodate disruptions, including those which result from lagging chromosomes, and achieve stereotypic outputs, such as proper chromosome separation.

High-resolution imaging reveals how the spindle midzone impacts chromosome movement.

In the spindle midzone, microtubules from opposite half-spindles form bundles between segregating chromosomes. Microtubule bundles can either push or restrict chromosome movement during anaphase in different cellular contexts, but how these activities are achieved remains poorly understood. Here, we use high-resolution live-cell imaging to analyze individual microtubule bundles, growing filaments, and chromosome movement in dividing human cells. Within bundles, filament overlap length marked by the cross-linking protein PRC1 decreases during anaphase as chromosome segregation slows. Filament ends within microtubule bundles appear capped despite dynamic PRC1 turnover and submicrometer proximity to growing microtubules. Chromosome segregation distance and rate are increased in two human cell lines when microtubule bundle assembly is prevented via PRC1 knockdown. Upon expressing a mutant PRC1 with reduced microtubule affinity, bundles assemble but chromosome hypersegregation is still observed. We propose that microtubule overlap length reduction, typically linked to pushing forces generated within filament bundles, is needed to properly restrict spindle elongation and position chromosomes within daughter cells.

The structured core of human ß tubulin confers isotype-specific polymerization properties.

Diversity in cytoskeleton organization and function may be achieved through variations in primary sequence of tubulin isotypes. Recently, isotype functional diversity has been linked to a "tubulin code" in which the C-terminal tail, a region of substantial sequence divergence between isotypes, specifies interactions with microtubule-associated proteins. However, it is not known whether residue changes in this region alter microtubule dynamic instability. Here, we examine recombinant tubulin with human ß isotype IIB and characterize polymerization dynamics. Microtubules with ßIIB have catastrophe frequencies approximately threefold lower than those with isotype ßIII, a suppression similar to that achieved by regulatory proteins. Further, we generate chimeric ß tubulins with native tail sequences swapped between isotypes. These chimeras have catastrophe frequencies similar to that of the corresponding full-length construct with the same core sequence. Together, our data indicate that residue changes within the conserved ß tubulin core are largely responsible for the observed isotype-specific changes in dynamic instability parameters and tune tubulin's polymerization properties across a wide range.

Mutations in Human Tubulin Proximal to the Kinesin-Binding Site Alter Dynamic Instability at Microtubule Plus- and Minus-Ends.

The assembly of microtubule-based cellular structures depends on regulated tubulin polymerization and directional transport. Here, we purify and characterize tubulin heterodimers that have human ß-tubulin isotype III (TUBB3), as well as heterodimers with one of two ß-tubulin mutations (D417H or R262H). Both point mutations are proximal to the kinesin-binding site and have been linked to an ocular motility disorder in humans. Compared to wild-type, microtubules with these mutations have decreased catastrophe frequencies and increased average lifetimes of plus- and minus-end-stabilizing caps. Importantly, the D417H mutation does not alter microtubule lattice structure or Mal3 binding to growing filaments. Instead, this mutation reduces the affinity of tubulin for TOG domains and colchicine, suggesting that the distribution of tubulin heterodimer conformations is changed. Together, our findings reveal how residues on the surface of microtubules, distal from the GTP-hydrolysis site and inter-subunit contacts, can alter polymerization dynamics at the plus- and minus-ends of microtubules.

A molecular evolution approach to study the roles of tropomyosin in fission yeast.

Tropomyosin, a coiled-coil protein that binds along the length of the actin filament, is a universal regulator of the actin cytoskeleton. We have taken a bioinformatics/proteomic approach to studying structure-function relationships in this protein. The presence of a single, essential tropomyosin gene, cdc8, in fission yeast, Schizosaccharomyces pombe, enables a systems-based approach to define the residues that are important for cellular functions. Using molecular evolution methodologies we identified the most conserved residues and related them to the coiled coil structure. Mutants in which one or more of 21 of the most conserved surface residues was mutated to Ala were tested for the ability to rescue growth of a temperature-sensitive cdc8 mutant when overexpressed at the restrictive temperature. Based on altered morphology of the septum and actin cytoskeleton, we selected three sets of mutations for construction of mutant cdc8 strains using marker reconstitution mutagenesis and analysis of recombinant protein in vitro: D16A.K30A, V114S.E117A.H118A and R121A.D131A.E138A. The mutations have sequence-specific effects on cellular morphology including cell length, organization of cytoskeletal structures (actin patches, actin cables and contractile rings), and in vitro actin affinity, lending credence to the proteomic approach introduced here. We propose that bioinformatics is a valid analysis tool for defining structure-function relationships in conserved proteins in this model organism.

Evolutionarily conserved surface residues constitute actin binding sites of tropomyosin.

Tropomyosin (Tm) is a two-chained, α-helical coiled-coil protein that associates end-to-end to form a continuous strand along actin filaments and regulates the functions and stability of actin in eukaryotic muscle and nonmuscle cells. Mutations in Tm cause skeletal and cardiac myopathies. We applied a neoteric molecular evolution approach to gain insight into the fundamental unresolved question of what makes the Tm coiled coil an actin binding protein. We carried out a phylogenetic analysis of 70 coding sequences of Tm genes from 26 animal species, from cnidarians to chordates, and evaluated the substitution rates (ω) at individual codons to identify conserved sites. The most conserved residues at surface b, c, f heptad repeat positions were mutated in rat striated muscle αTm and expressed in Escherichia coli. Each mutant had 3-4 sites mutated to Ala within the first half or the second half of periods 2-6. Actin affinity and thermodynamic stability were determined in vitro. Mutations in the first half of periods 2, 4, and 5 resulted in the largest reduction in actin affinity (> 4-fold), indicating these mutations include residues in actin-binding sites. Mutations in the second half of the periods had a ≤ 2-fold effect on affinity indicating these residues may be involved in other conserved regulatory functions. The structural relevance of these results was assessed by constructing molecular models for the actin-Tm filament. Molecular evolution analysis is a general approach that may be used to identify potential binding sites of a protein for a conserved protein.