The Clathrin-dependent Spindle Proteome.

The mitotic spindle is required for chromosome congression and subsequent equal segregation of sister chromatids. These processes involve a complex network of signaling molecules located at the spindle. The endocytic protein, clathrin, has a "moonlighting" role during mitosis, whereby it stabilizes the mitotic spindle. The signaling pathways that clathrin participates in to achieve mitotic spindle stability are unknown. Here, we assessed the mitotic spindle proteome and phosphoproteome in clathrin-depleted cells using quantitative MS/MS (data are available via ProteomeXchange with identifier PXD001603). We report a spindle proteome that consists of 3046 proteins and a spindle phosphoproteome consisting of 5157 phosphosites in 1641 phosphoproteins. Of these, 2908 (95.4%) proteins and 1636 (99.7%) phosphoproteins are known or predicted spindle-associated proteins. Clathrin-depletion from spindles resulted in dysregulation of 121 proteins and perturbed signaling to 47 phosphosites. The majority of these proteins increased in mitotic spindle abundance and six of these were validated by immunofluorescence microscopy. Functional pathway analysis confirmed the reported role of clathrin in mitotic spindle stabilization for chromosome alignment and highlighted possible new mechanisms of clathrin action. The data also revealed a novel second mitotic role for clathrin in bipolar spindle formation.

A Cholesterol-Dependent Endocytic Mechanism Generates Midbody Tubules During Cytokinesis.

Cytokinesis is the final stage of cell division and produces two independent daughter cells. Vesicles derived from internal membrane stores, such as the Golgi, lysosomes, and early and recycling endosomes accumulate at the intracellular bridge (ICB) during cytokinesis. Here, we use electron tomography to show that many ICB vesicles are not independent but connected, forming a newly described ICB vesicular structure - narrow tubules that are often branched. These 'midbody tubules' labelled with horseradish peroxidase (HRP) within 10 min after addition to the surrounding medium demonstrating that they are derived from endocytosis. HRP-labelled vesicles and tubules were observed at the rim of the ICB after only 1 min, suggesting that midbody tubules are likely to be generated by local endocytosis occurring at the ICB rim. Indeed, at least one tubule was open to the extracellular space, indicative of a local origin within the ICB. Inhibition of cholesterol-dependent endocytosis by exposure to methyl-ß-cyclodextrin and filipin reduced formation of HRP-labelled midbody tubules, and induced multinucleation following ICB formation. In contrast, dynamin inhibitors, which block clathrin-mediated endocytosis, induced multinucleation but had no effect on the formation of HRP-labelled midbody tubules. Therefore, our data reveal the existence of a cholesterol-dependent endocytic pathway occurring locally at the ICB, which contributes to the accumulation of vesicles and tubules that contribute to the completion of cytokinesis.

Electrokinetic properties of the mammalian tectorial membrane.

The tectorial membrane (TM) clearly plays a mechanical role in stimulating cochlear sensory receptors, but the presence of fixed charge in TM constituents suggests that electromechanical properties also may be important. Here, we measure the fixed charge density of the TM and show that this density of fixed charge is sufficient to affect mechanical properties and to generate electrokinetic motions. In particular, alternating currents applied to the middle and marginal zones of isolated TM segments evoke motions at audio frequencies (1-1,000 Hz). Electrically evoked motions are nanometer scaled (∼5-900 nm), decrease with increasing stimulus frequency, and scale linearly over a broad range of electric field amplitudes (0.05-20 kV/m). These findings show that the mammalian TM is highly charged and suggest the importance of a unique TM electrokinetic mechanism.

Calcineurin activity is required for the completion of cytokinesis.

Successful completion of cytokinesis requires the spatio-temporal regulation of protein phosphorylation and the coordinated activity of protein kinases and phosphatases. Many mitotic protein kinases are well characterized while mitotic phosphatases are largely unknown. Here, we show that the Ca(2+)- and calmodulin-dependent phosphatase, calcineurin (CaN), is required for cytokinesis in mammalian cells, functioning specifically at the abscission stage. CaN inhibitors induce multinucleation in HeLa cells and prolong the time cells spend connected via an extended intracellular bridge. Upon Ca(2+) influx during cytokinesis, CaN is activated, targeting a set of proteins for dephosphorylation, including dynamin II (dynII). At the intracellular bridge, phospho-dynII and CaN are co-localized to dual flanking midbody rings (FMRs) that reside on either side of the central midbody ring. CaN activity and disassembly of the FMRs coincide with abscission. Thus, CaN activity at the midbody plays a key role in regulating the completion of cytokinesis in mammalian cells.

Corona is required for higher-order assembly of transverse filaments into full-length synaptonemal complex in Drosophila oocytes.

The synaptonemal complex (SC) is an intricate structure that forms between homologous chromosomes early during the meiotic prophase, where it mediates homolog pairing interactions and promotes the formation of genetic exchanges. In Drosophila melanogaster, C(3)G protein forms the transverse filaments (TFs) of the SC. The N termini of C(3)G homodimers localize to the Central Element (CE) of the SC, while the C-termini of C(3)G connect the TFs to the chromosomes via associations with the axial elements/lateral elements (AEs/LEs) of the SC. Here, we show that the Drosophila protein Corona (CONA) co-localizes with C(3)G in a mutually dependent fashion and is required for the polymerization of C(3)G into mature thread-like structures, in the context both of paired homologous chromosomes and of C(3)G polycomplexes that lack AEs/LEs. Although AEs assemble in cona oocytes, they exhibit defects that are characteristic of c(3)G mutant oocytes, including failure of AE alignment and synapsis. These results demonstrate that CONA, which does not contain a coiled coil domain, is required for the stable 'zippering' of TFs to form the central region of the Drosophila SC. We speculate that CONA's role in SC formation may be similar to that of the mammalian CE proteins SYCE2 and TEX12. However, the observation that AE alignment and pairing occurs in Tex12 and Syce2 mutant meiocytes but not in cona oocytes suggests that the SC plays a more critical role in the stable association of homologs in Drosophila than it does in mammalian cells.

The Drosophila cohesin subunit Rad21 is a trithorax group (trxG) protein.

The cohesin complex is a key player in regulating cell division. Cohesin proteins SMC1, SMC3, Rad21, and stromalin (SA), along with associated proteins Nipped-B, Pds5, and EcoI, maintain sister chromatid cohesion before segregation to daughter cells during anaphase. Recent chromatin immunoprecipitation (ChIP) data reveal extensive overlap of Nipped-B and cohesin components with RNA polymerase II binding at active genes in Drosophila. These and other data strongly suggest a role for cohesion in transcription; however, there is no clear evidence for any specific mechanisms by which cohesin and associated proteins regulate transcription. We report here a link between cohesin components and trithorax group (trxG) function, thus implicating these proteins in transcription activation and/or elongation. We show that the Drosophila Rad21 protein is encoded by verthandi (vtd), a member of the trxG gene family that is also involved in regulating the hedgehog (hh) gene. In addition, mutations in the associated protein Nipped-B show similar trxG activity i.e., like vtd, they act as dominant suppressors of Pc and hh(Mrt) without impairing cell division. Our results provide a framework to further investigate how cohesin and associated components might regulate transcription.

The formation of the central element of the synaptonemal complex may occur by multiple mechanisms: the roles of the N- and C-terminal domains of the Drosophila C(3)G protein in mediating synapsis and recombination.

In Drosophila melanogaster oocytes, the C(3)G protein comprises the transverse filaments (TFs) of the synaptonemal complex (SC). Like other TF proteins, such as Zip1p in yeast and SCP1 in mammals, C(3)G is composed of a central coiled-coil-rich domain flanked by N- and C-terminal globular domains. Here, we analyze in-frame deletions within the N- and C-terminal regions of C(3)G in Drosophila oocytes. As is the case for Zip1p, a C-terminal deletion of C(3)G fails to attach to the lateral elements of the SC. Instead, this C-terminal deletion protein forms a large cylindrical polycomplex structure. EM analysis of this structure reveals a polycomplex of concentric rings alternating dark and light bands. However, unlike both yeast and mammals, all three proteins deleted for N-terminal regions completely abolished both SC and polycomplex formation. Both the N- and C-terminal deletions significantly reduce or abolish meiotic recombination similarly to c(3)G null homozygotes. To explain these data, we propose that in Drosophila the N terminus, but not the C-terminal globular domain, of C(3)G is critical for the formation of antiparallel pairs of C(3)G homodimers that span the central region and thus for assembly of complete TFs, while the C terminus is required to affix these homodimers to the lateral elements.

A genetic analysis of the Drosophila mcm5 gene defines a domain specifically required for meiotic recombination.

Members of the minichromosome maintenance (MCM) family have pivotal roles in many biological processes. Although originally studied for their role in DNA replication, it is becoming increasingly apparent that certain members of this family are multifunctional and also play roles in transcription, cohesion, condensation, and recombination. Here we provide a genetic dissection of the mcm5 gene in Drosophila that demonstrates an unexpected function for this protein. First, we show that homozygotes for a null allele of mcm5 die as third instar larvae, apparently as a result of blocking those replication events that lead to mitotic divisions without impairing endo-reduplication. However, we have also recovered a viable and fertile allele of mcm5 (denoted mcm5(A7)) that specifically impairs the meiotic recombination process. We demonstrate that the decrease in recombination observed in females homozygous for mcm5(A7) is not due to a failure to create or repair meiotically induced double strand breaks (DSBs), but rather to a failure to resolve those DSBs into meiotic crossovers. Consistent with their ability to repair meiotically induced DSBs, flies homozygous for mcm5(A7) are fully proficient in somatic DNA repair. These results strengthen the observation that members of the prereplicative complex have multiple functions and provide evidence that mcm5 plays a critical role in the meiotic recombination pathway.

A germline clone screen for meiotic mutants in Drosophila melanogaster.

Using an FLP/FRT-based method to create germline clones, we screened Drosophila chromosome arms 2L and 3R for new female meiotic mutants. The screen was designed to recover mutants with severe effects on meiotic exchange and/or segregation. This screen yielded 11 new mutants, including six alleles of previously known meiotic genes (c(2)M and ald/mps1). The remaining five mutants appear to define at least four new genes whose ablation results in severe meiotic defects. Three of the novel meiotic mutants were identified at the molecular level. Two of these, mcm5(A7) and trem(F9), define roles in meiotic recombination, while a third, cona(A12), is important for synaptonemal complex assembly. Surprisingly, five of the nine mutants for which the lesion has been identified at the molecular level are not the result of mutations characteristic of EMS mutagenesis, but rather due to the insertion of the transposable element Doc. This study demonstrates the utility of germline clone-based screens for the discovery of strong meiotic mutants, including mutations in essential genes, and the use of molecular genetic techniques to map the loci.

The Drosophila meiotic mutant mei-352 is an allele of klp3A and reveals a role for a kinesin-like protein in crossover distribution.

The semisterile meiotic mutant mei-352 alters the distribution of meiotic exchanges without greatly affecting their total frequency. We show that the mei-352 mutation is an allele of the klp3A gene, which encodes a kinesin-like protein of the Kinesin-4 family. The semisterility observed in mei-352 females results from a known defect of klp3A oocytes in mediating pronuclear fusion. Interestingly, other klp3A alleles also exhibit defects in meiotic recombination similar to those of mei-352. Finally, we show that the Klp3A protein localizes within the oocyte nucleus during meiotic prophase, the time at which exchange distribution is established, and extensively colocalizes with DNA. The parallel of the klp3A phenotype with a meiotic defect observed for kar3 mutants in yeast suggests a role for kinesins in early meiosis and might reflect a previously suggested role for this class of kinesins in chromosome condensation.

Juxtaposition of C(2)M and the transverse filament protein C(3)G within the central region of Drosophila synaptonemal complex.

The synaptonemal complex (SC) is intimately involved in the process of meiotic recombination in most organisms, but its exact role remains enigmatic. One reason for this uncertainty is that the overall structure of the SC is evolutionarily conserved, but many SC proteins are not. Two putative SC proteins have been identified in Drosophila: C(3)G and C(2)M. Mutations in either gene cause defects in SC structure and meiotic recombination. Although neither gene is well conserved at the amino acid level, the predicted secondary structure of C(3)G is similar to that of transversefilament proteins, and C(2)M is a distantly related member of the alpha-kleisin family that includes Rec8, a meiosis-specific cohesin protein. Here, we use immunogold labeling of SCs in Drosophila ovaries to localize C(3)G and C(2)M at the EM level. We show that both C(3)G and C(2)M are components of the SC, that the orientation of C(3)G within the SC is similar to other transverse-filament proteins, and that the N terminus of C(2)M is located in the central region adjacent to the lateral elements (LEs). Based on our data and the known phenotypes of C(2)M and C(3)G mutants, we propose a model of SC structure in which C(2)M links C(3)G to the LEs.

The genetics and molecular biology of the synaptonemal complex.

The synaptonemal complex (SC) is a protein lattice that resembles railroad tracks and connects paired homologous chromosomes in most meiotic systems. The two side rails of the SC, known as lateral elements (LEs), are connected by proteins known as transverse filaments. The LEs are derived from the axial elements of the chromosomes and play important roles in chromosome condensation, pairing, transverse filament assembly, and prohibiting double-strand breaks (DSBs) from entering into recombination pathways that involve sister chromatids. The proteins that make up the transverse filaments of the SC also play a much earlier role in committing a subset of DSBs into a recombination pathway, which results in the production of reciprocal meiotic crossovers. Sites of crossover commitment can be observed as locations where the SC initiates and as immunostaining foci for a set of proteins required for the processing of DSBs to mature crossovers. In most (but not all) organisms it is the establishment of sites marking such crossover-committed DSBs that facilitates completion of synapsis (full-length extension of the SC). The function of the mature full-length SC may involve both the completion of meiotic recombination at the DNA level and the exchange of the axial elements of the two chromatids involved in the crossover. However, the demonstration that the sites of crossover formation are designated prior to SC formation, and the finding that these sites display interference, argues against a role of the mature SC in mediating the process of interference. Finally, in at least some organisms, modifications of the SC alone are sufficient to ensure meiotic chromosome segregation in the complete absence of meiotic recombination.

Chromosome choreography: the meiotic ballet.

The separation of homologous chromosomes during meiosis in eukaryotes is the physical basis of Mendelian inheritance. The core of the meiotic process is a specialized nuclear division (meiosis I) in which homologs pair with each other, recombine, and then segregate from each other. The processes of chromosome alignment and pairing allow for homolog recognition. Reciprocal meiotic recombination ensures meiotic chromosome segregation by converting sister chromatid cohesion into mechanisms that hold homologous chromosomes together. Finally, the ability of sister kinetochores to orient to a single pole at metaphase I allows the separation of homologs to two different daughter cells. Failures to properly accomplish this elegant chromosome dance result in aneuploidy, a major cause of miscarriage and birth defects in human beings.

Obligate short-arm exchange in de novo Robertsonian translocation formation influences placement of crossovers in chromosome 21 nondisjunction.

Robertsonian translocations (ROBs) involving chromosome 21 are found in approximately 5% of patients with Down syndrome (DS). The most common nonhomologous ROB in DS is rob(14q21q). Aberrant recombination is associated with nondisjunction (NDJ) leading to trisomy 21. Haplotype analysis of 23 patients with DS and de novo rob(14q21q) showed that all translocations and all nondisjoined chromosomes 21 were maternally derived. Meiosis II NDJ occurred in 21 of 23 families. For these, a ROB DS chromosome 21 genetic map was constructed and compared to a normal female map and a published trisomy 21 map derived from meiosis II NDJ. The location of exchanges differed significantly from both maps, with a significant shift to a more distal interval in the ROB DS map. The shift may perturb segregation, leading to the meiosis II NDJ in this study, and is further evidence for crossover interference. More importantly, because the event in the short arms that forms the de novo ROB influences the placement of chiasmata in the long arm, it is most likely that the translocation formation occurs through a recombination pathway in meiosis. Additionally, we have demonstrated that events that occur in meiosis I can influence events, such as chromatid segregation in meiosis II, many decades later.

Parental origin and timing of de novo Robertsonian translocation formation.

Robertsonian translocations (ROBs) are the most common chromosomal rearrangements in humans. ROBs are whole-arm rearrangements between the acrocentric chromosomes 13-15, 21, and 22. ROBs can be classified into two groups depending on their frequency of occurrence, common (rob(13q14q) and rob(14q21q)), and rare (all remaining possible nonhomologous combinations). Herein, we have studied 29 case subjects of common and rare de novo ROBs to determine their parental origins and timing of formation. We compared these case subjects to 35 published case subjects of common ROBs and found that most common ROBs apparently have the same breakpoints and arise mainly during oogenesis (50/54). These probably form through a common mechanism and have been termed "class 1." Collectively, rare ROBs also occur mostly during oogenesis (7/10) but probably arise through a more "random" mechanism or a variety of mechanisms and have been termed "class 2." Thus, we demonstrate that although both classes of ROBs occur predominantly during meiosis, the common, class 1 ROBs occur primarily during oogenesis and likely form through a mechanism distinct from that forming class 2 ROBs.