Here we probe the relationships between assembly of the synaptonemal complex (SC) and progression of recombination between homologous chromosomes during Caenorhabditis elegans meiosis. We identify SYP-2 as a structural component of the SC central region and show that central region assembly depends on proper morphogenesis of chromosome axes. We find that the SC central region is dispensable for initiation of recombination and for loading of DNA strand-exchange protein RAD-51, despite the fact that extensive RAD-51 loading normally occurs in the context of assembled SC. Further, persistence of RAD-51 foci and absence of crossover products in meiotic mutants suggests that SC central region components and recombination proteins MSH-4 and MSH-5 are required to promote conversion of resected double-strand breaks into stable post-strand exchange intermediates. Our data also suggest that early prophase barriers to utilization of sister chromatids as repair templates do not depend on central region assembly.

The protein folding capacity of the endoplasmic reticulum (ER) is regulated by the unfolded protein response (UPR). The UPR senses unfolded proteins in the ER lumen and transmits that information to the cell nucleus, where it drives a transcriptional program that is tailored to re-establish homeostasis. Using thin section electron microscopy, we found that yeast cells expand their ER volume at least 5-fold under UPR-inducing conditions. Surprisingly, we discovered that ER proliferation is accompanied by the formation of autophagosome-like structures that are densely and selectively packed with membrane stacks derived from the UPR-expanded ER. In analogy to pexophagy and mitophagy, which are autophagic processes that selectively sequester and degrade peroxisomes and mitochondria, the ER-specific autophagic process described utilizes several autophagy genes: they are induced by the UPR and are essential for the survival of cells subjected to severe ER stress. Intriguingly, cell survival does not require vacuolar proteases, indicating that ER sequestration into autophagosome-like structures, rather than their degradation, is the important step. Selective ER sequestration may help cells to maintain a new steady-state level of ER abundance even in the face of continuously accumulating unfolded proteins.

Chromosome segregation at meiosis I depends on pairing and crossing-over between homologs. In most eukaryotes, pairing culminates with formation of the proteinaceous synaptonemal complex (SC). In budding yeast, recombination initiates through double-strand DNA breaks (DSBs) and is thought to be essential for SC formation. Here, we examine whether this mechanism for initiating meiotic recombination is conserved, and we test the dependence of homologous chromosome synapsis on recombination in C. elegans. We find that a homolog of the yeast DSB-generating enzyme, Spo11p, is required for meiotic exchange in this metazoan, and that radiation-induced breaks partially alleviate this dependence. Thus, initiation of recombination by DSBs is apparently conserved. However, homologous synapsis is independent of recombination in the nematode, since it occurs normally in a C. elegans spo-11 null mutant.

Analysis of Caenorhabditis elegans syp-1 mutants reveals that both synapsis-dependent and -independent mechanisms contribute to stable, productive alignment of homologous chromosomes during meiotic prophase. Early prophase nuclei undergo normal reorganization in syp-1 mutants, and chromosomes initially pair. However, the polarized nuclear organization characteristic of early prophase persists for a prolonged period, and homologs dissociate prematurely; furthermore, the synaptonemal complex (SC) is absent. The predicted structure of SYP-1, its localization at the interface between intimately paired, lengthwise-aligned pachytene homologs, and its kinetics of localization with chromosomes indicate that SYP-1 is an SC structural component. A severe reduction in crossing over together with evidence for accumulated recombination intermediates in syp-1 mutants indicate that initial pairing is not sufficient for completion of exchange and implicates the SC in promoting crossover recombination. Persistence of polarized nuclear organization in syp-1 mutants suggests that SC polymerization may provide a motive force or signal that drives redispersal of chromosomes. Whereas our analysis suggests that the SC is required to stabilize pairing along the entire lengths of chromosomes, striking differences in peak pairing levels for opposite ends of chromosomes in syp-1 mutants reveal the existence of an additional mechanism that can promote local stabilization of pairing, independent of synapsis. At the onset of meiosis, an extensive spatial reorganization of chromosomes within the nucleus culminates in an arrangement in which homologous chromosomes are lengthwise-aligned, intimately paired, and capable of undergoing crossover recombination. For nearly all sexually reproducing organisms, the accuracy of chromosome segregation during meiosis I depends on the physical exchange between DNA molecules of homologous chromosomes provided by crossover recombination events that are completed in this context. In conjunction with sister-chromatid cohesion, crossing over results in the formation of chiasmata, which serve as mechanical connections that facilitate proper orientation and subsequent segregation of homologs toward opposite poles of the meiosis I spindle (Roeder 1997;Zickler and Kleckner 1999).Specific associations between homologs are established early in meiotic prophase and are maintained prior to and during completion of crossover recombination and chiasma formation. Initial pairing events occur soon after premeiotic S phase, and are typically accompanied by an overall spatial reorganization of the nucleus that leads to a striking polarized distribution of chromosomes and other nuclear contents (for reviews, see Zickler and Kleckner 1998;Scherthan 2001). Many or all aspects of early prophase polarization are lost upon entry into the pachytene stage of meiotic prophase, as homologous chromosomes achieve full intimate alignment with one another along their entire lengths. A hallmark feature of pachytene chromosome organization ...

Abstract. The three dimensional organization of microtubules in mitotic spindles of the yeast Saccharomyces cerevisiae has been determined by computer-aided reconstruction from electron micrographs of serially cross-sectioned spindles. Fifteen spindles ranging in length from 0.6-9.4 Ixm have been analyzed. Ordered microtubule packing is absent in spindles up to 0.8 Ixm, but the total number of microtubules is sufficient to allow one microtubule per kinetochore with a few additional microtubules that may form an interpolar spindle. An obvious bundle of about eight interpolar microtubules was found in spindles 1.3-1.6 p~m long, and we suggest that the ~32 remaining microtubules act as kinetochore fibers. The relative lengths of the microtubules in these spindles suggest that they may be in an early stage of anaphase, even though these spindles are all situated in the mother cell, not in the isthmus between mother and bud. None of the reconstructed spindles exhibited the uniform populations of kinetochore microtubules characteristic of metaphase. Long spindles (2.7-9.4 p,m), presumably in anaphase B, contained short remnants of a few presumed kinetochore microtubules clustered near the poles and a few long microtubules extending from each pole toward the spindle midplane, where they interdigitated with their counterparts from the other pole. Interpretation of these reconstructed spindles offers some insights into the mechanisms of mitosis in this yeast. THE structure of the mitotic spindle and its function have been studied in a wide variety of organisms, yielding a few global observations about spindle structure. In general, spindles are organized from two spindle poles, each of which nucleates several classes of microtubules. These classes include astral, kinetochore, and interpolar spindle microtubules. The astral microtubules are not part of the spindle per se, but can be involved in the orientation, and perhaps the elongation of the spindle. Kinetochore microtubules connect the chromosomes to the spindle poles and are involved in chromosome movements. The interpolar spindle microtubules extend from each spindle pole, interdigitate with their counterparts from the other pole, and are involved in separating the poles with their attached chromosomes during anaphase B. This generic view of spindle organization is thought by many to hold true for most spindles, including

As the main microtubule-organizing center in animal cells, the centrosome has a fundamental role in cell function. Surrounding the centrioles, the Pericentriolar material (PCM) provides a dynamic platform for nucleating microtubules. While the PCM’s importance is established, its amorphous electron-dense nature has made it refractory to structural investigation. By using SIM and STORM sub-diffraction resolution microscopies to visualize proteins critical for centrosome maturation, we demonstrate that the PCM is organized into two major structural domains: a layer juxtaposed to the centriole wall, and proteins extending further away from the centriole organized in a matrix. Analysis of Pericentrin-like protein (Plp) reveals that its C-terminus is positioned at the centriole wall, it radiates outward into the matrix and is organized in clusters having quasi-nine-fold symmetry. By RNAi we show that Plp fibrils are required for interphase recruitment and proper mitotic assembly of the PCM matrix.

It has been posited that animal development evolved from pre-existing mechanisms for regulating cell differentiation in the single celled and colonial ancestors of animals. Although the progenitors of animals cannot be studied directly, insights into their cell biology may be gleaned from comparisons between animals and their closest living relatives, the choanoflagellates. We report here on the life history, cell differentiation and intercellular interactions in the colony-forming choanoflagellate Salpingoeca rosetta. In response to diverse environmental cues, S. rosetta differentiates into at least five distinct cell types, including three solitary cell types (slow swimmers, fast swimmers, and thecate cells) and two colonial forms (rosettes and chains). Electron microscopy reveals that cells within colonies are held together by a combination of fine intercellular bridges, a shared extracellular matrix, and filopodia. In addition, we have discovered that the carbohydrate-binding protein wheat germ agglutinin specifically stains colonies and the slow swimmers from which they form, showing that molecular differentiation precedes multicellular development. Together, these results help establish S. rosetta as a model system for studying simple multicellularity in choanoflagellates and provide an experimental framework for investigating the origin of animal multicellularity and development.

Previous genetic and biochemical studies have led to the hypothesis that the essential mitotic bipolar kinesin, KLP61F, cross-links and slides microtubules (MTs) during spindle assembly and function. Here, we have tested this hypothesis by immunofluorescence and immunoelectron microscopy (immunoEM). We show that Drosophila embryonic spindles at metaphase and anaphase contain abundant bundles of MTs running between the spindle poles. These interpolar MT bundles are parallel near the poles and antiparallel in the midzone. We have observed that KLP61F motors, phosphorylated at a cdk1/cyclin B consensus domain within the BimC box (BCB), localize along the length of these interpolar MT bundles, being concentrated in the midzone region. Nonphosphorylated KLP61F motors, in contrast, are excluded from the spindle and display a cytoplasmic localization. Immunoelectron microscopy further suggested that phospho-KLP61F motors form cross-links between MTs within interpolar MT bundles. These bipolar KLP61F MT-MT cross-links should be capable of organizing parallel MTs into bundles within half spindles and sliding antiparallel MTs apart in the spindle midzone. Thus we propose that bipolar kinesin motors and MTs interact by a “sliding filament mechanism” during the formation and function of the mitotic spindle.