Live-imaging analysis of germ cell proliferation in the C. elegans adult supports a stochastic model for stem cell proliferation.

The C. elegans adult hermaphrodite contains a renewable pool of mitotically dividing germ cells that are contained within the progenitor zone (PZ), at the distal region of the germline. From the PZ, cells enter meiosis and differentiate, ensuring the continued production of oocytes. In this study, we investigated the proliferation strategy used to maintain the PZ pool by using a photoconvertible marker to follow the fate of selected germ cells and their descendants in live worms. We found that the most distal pool of 6-8 rows of cells in the PZ (the distal third) behave similarly, with a fold expansion corresponding to one cell division every 6h on average. Proximal to this region, proliferation decreases, and by the proximal third of the PZ, most cells have stopped dividing. In addition, we show that all the descendants of cells in rows 3 and above move proximally and leave the PZ over time. Combining our data with previous studies, we propose a stochastic model for the C. elegans PZ proliferation, where a pool of proliferating stem cells divide symmetrically within the distal most 6-8 rows of the germline and exit from this stem cell niche occurs by displacement due to competition for limited space.

Down-regulation of tricarboxylic acid (TCA) cycle genes blocks progression through the first mitotic division in Caenorhabditis elegans embryos.

The cell cycle is a highly regulated process that enables the accurate transmission of chromosomes to daughter cells. Here we uncover a previously unknown link between the tricarboxylic acid (TCA) cycle and cell cycle progression in the Caenorhabditis elegans early embryo. We found that down-regulation of TCA cycle components, including citrate synthase, malate dehydrogenase, and aconitase, resulted in a one-cell stage arrest before entry into mitosis: pronuclear meeting occurred normally, but nuclear envelope breakdown, centrosome separation, and chromosome condensation did not take place. Mitotic entry is controlled by the cyclin B-cyclin-dependent kinase 1 (Cdk1) complex, and the inhibitory phosphorylation of Cdk1 must be removed in order for the complex to be active. We found that following down-regulation of the TCA cycle, cyclin B levels were normal but CDK-1 remained inhibitory-phosphorylated in one-cell stage-arrested embryos, indicative of a G2-like arrest. Moreover, this was not due to an indirect effect caused by checkpoint activation by DNA damage or replication defects. These observations suggest that CDK-1 activation in the C. elegans one-cell embryo is sensitive to the metabolic state of the cell, and that down-regulation of the TCA cycle prevents the removal of CDK-1 inhibitory phosphorylation. The TCA cycle was previously shown to be necessary for the development of the early embryo in mammals, but the molecular processes affected were not known. Our study demonstrates a link between the TCA cycle and a specific cell cycle transition in the one-cell stage embryo.

The Malleable Nature of the Budding Yeast Nuclear Envelope: Flares, Fusion, and Fenestrations.

In eukaryotes, the nuclear envelope (NE) physically separates nuclear components and activities from rest of the cell. The NE also provides rigidity to the nucleus and contributes to chromosome organization. At the same time, the NE is highly dynamic; it must change shape and rearrange its components during development and throughout the cell cycle, and its morphology can be altered in response to mutation and disease. Here we focus on the NE of budding yeast, Saccharomyces cerevisiae, which has several unique features: it remains intact throughout the cell cycle, expands symmetrically during interphase, elongates during mitosis and, expands asymmetrically during mitotic delay. Moreover, its NE is safely breached during mating and when large structures, such as nuclear pore complexes and the spindle pole body, are embedded into its double membrane. The budding yeast NE lacks lamins and yet the nucleus is capable of maintaining a spherical shape throughout interphase. Despite these eccentricities, studies of the budding yeast NE have uncovered interesting, and likely conserved, processes that contribute to NE dynamics. In particular, we discuss the processes that drive and enable NE expansion and the dramatic changes in the NE that lead to extensions and fenestrations. J. Cell. Physiol. 231: 2353-2360, 2016. Published 2016. This article is a U.S. Government work and is in the public domain in the USA.

Putting molecules in their place.

Each class of microscope is limited to imaging specific aspects of cell structure and/or molecular organization. However, imaging the specimen by complementary microscopes and correlating the data can overcome this limitation. Whilst not a new approach, the field of correlative imaging is currently benefitting from the emergence of new microscope techniques. Here we describe the correlation of cryogenic fluorescence tomography (CFT) with soft X-ray tomography (SXT). This amalgamation of techniques integrates 3D molecular localization data (CFT) with a high-resolution, 3D cell reconstruction of the cell (SXT). Cells are imaged in both modalities in a near-native, cryopreserved state. Here we describe the current state of the art in correlative CFT-SXT, and discuss the future outlook for this method.

Sm protein down-regulation leads to defects in nuclear pore complex disassembly and distribution in C. elegans embryos.

Nuclear pore complexes (NPCs) are large macromolecular structures embedded in the nuclear envelope (NE), where they facilitate exchange of molecules between the cytoplasm and the nucleoplasm. In most cell types, NPCs are evenly distributed around the NE. However, the mechanisms dictating NPC distribution are largely unknown. Here, we used the model organism Caenorhabditis elegans to identify genes that affect NPC distribution during early embryonic divisions. We found that down-regulation of the Sm proteins, which are core components of the spliceosome, but not down-regulation of other splicing factors, led to clustering of NPCs. Down-regulation of Sm proteins also led to incomplete disassembly of NPCs during mitosis, but had no effect on lamina disassembly, suggesting that the defect in NPC disassembly was not due to a general defect in nuclear envelope breakdown. We further found that these mitotic NPC remnants persisted on an ER membrane that juxtaposes the mitotic spindle. At the end of mitosis, the remnant NPCs moved toward the chromatin and the reforming NE, where they ultimately clustered by forming membrane stacks perforated by NPCs. Our results suggest a novel, splicing-independent, role for Sm proteins in NPC disassembly, and point to a possible link between NPC disassembly in mitosis and NPC distribution in the subsequent interphase.

A membrane reticulum, the centriculum, affects centrosome size and function in Caenorhabditis elegans.

Centrosomes are cellular structures that nucleate microtubules. At their core is a pair of centrioles that recruit pericentriolar material (PCM). Although centrosomes are considered membraneless organelles, in many cell types, including human cells, centrosomes are surrounded by endoplasmic reticulum (ER)-derived membranes of unknown structure and function. Using volume electron microscopy (vEM), we show that centrosomes in the Caenorhabditis elegans (C. elegans) early embryo are surrounded by a three-dimensional (3D), ER-derived membrane reticulum that we call the centriculum, for centrosome-associated membrane reticulum. The centriculum is adjacent to the nuclear envelope in interphase and early mitosis and fuses with the fenestrated nuclear membrane at metaphase. Centriculum formation is dependent on the presence of an underlying centrosome and on microtubules. Conversely, increasing centriculum size by genetic means led to the expansion of the PCM, increased microtubule nucleation capacity, and altered spindle width. The effect of the centriculum on centrosome function suggests that in the C. elegans early embryo, the centrosome is not membraneless. Rather, it is encased in a membrane meshwork that affects its properties. We provide evidence that the centriculum serves as a microtubule "filter," preventing the elongation of a subset of microtubules past the centriculum. Finally, we propose that the fusion between the centriculum and the nuclear membrane contributes to nuclear envelope breakdown by coupling spindle elongation to nuclear membrane fenestration.

Shaping the nucleus: factors and forces.

Take a look at a textbook illustration of a cell and you will immediately be able to locate the nucleus, which is often drawn as a spherical or ovoid shaped structure. But not all cells have such nuclei. In fact, some disease states are diagnosed by the presence of nuclei that have an abnormal shape or size. What defines nuclear shape and nuclear size, and how does nuclear geometry affect nuclear function? While the answer to the latter question remains largely unknown, significant progress has been made towards understanding the former. In this review, we provide an overview of the factors and forces that affect nuclear shape and size, discuss the relationship between ER structure and nuclear morphology, and speculate on the possible connection between nuclear size and its shape. We also note the many interesting questions that remain to be explored.

Cell biology: How does the nucleus get its membrane?

Nuclear shape and size depend on nuclear membrane availability through an unknown process. A new study of asymmetric cell division reveals that nuclear membrane is derived from the endoplasmic reticulum and that limiting nuclear membrane expansion can affect cell fate.

An RNAi screen for genes that affect nuclear morphology in Caenorhabditis elegans reveals the involvement of unexpected processes.

Aberration in nuclear morphology is one of the hallmarks of cellular transformation. However, the processes that, when mis-regulated, result aberrant nuclear morphology are poorly understood. In this study, we carried out a systematic, high-throughput RNAi screen for genes that affect nuclear morphology in Caenorhabditis elegans embryos. The screen employed over 1700 RNAi constructs against genes required for embryonic viability. Nuclei of early embryos are typically spherical, and their NPCs are evenly distributed. The screen was performed on early embryos expressing a fluorescently tagged component of the nuclear pore complex (NPC), allowing visualization of nuclear shape as well as the distribution of NPCs around the nuclear envelope. Our screen uncovered 182 genes whose downregulation resulted in one or more abnormal nuclear phenotypes, including multiple nuclei, micronuclei, abnormal nuclear shape, anaphase bridges, and abnormal NPC distribution. Many of these genes fall into common functional groups, including some that were not previously known to affect nuclear morphology, such as genes involved in mitochondrial function, the vacuolar ATPase, and the CCT chaperonin complex. The results of this screen add to our growing knowledge of processes that affect nuclear morphology and that may be altered in cancer cells that exhibit abnormal nuclear shape.

A Workflow for High-pressure Freezing and Freeze Substitution of the Caenorhabditis elegans Embryo for Ultrastructural Analysis by Conventional and Volume Electron Microscopy.

The free-living nematode Caenorhabditis elegans is a popular model system for studying developmental biology. Here we describe a detailed protocol to high-pressure freeze the C. elegans embryo (either ex vivo after dissection, or within the intact worm) followed by quick freeze substitution. Processed samples are suitable for ultrastructural analysis by conventional electron microscopy (EM) or newer volume EM (vEM) approaches such as Focused Ion Beam Scanning Electron Microscopy (FIB-SEM). The ultrastructure of cellular features such as the nuclear envelope, chromosomes, endoplasmic reticulum and mitochondria are well preserved after these experimental procedures and yield accurate 3D models for visualization and analysis ( Chang et al., 2020 ). This protocol was used in the 3D reconstruction of membranes and chromosomes after pronuclear meeting in the C. elegans zygote ( Rahman et al., 2020 ).

Cryo-fluorescence microscopy of high-pressure frozen C. elegans enables correlative FIB-SEM imaging of targeted embryonic stages in the intact worm.

Rapidly changing features in an intact biological sample are challenging to efficiently trap and image by conventional electron microscopy (EM). For example, the model organism C. elegans is widely used to study embryonic development and differentiation, yet the fast kinetics of cell division makes the targeting of specific developmental stages for ultrastructural study difficult. We set out to image the condensed metaphase chromosomes of an early embryo in the intact worm in 3-D. To achieve this, one must capture this transient structure, then locate and subsequently image the corresponding volume by EM in the appropriate context of the organism, all while minimizing a variety of artifacts. In this methodological advance, we report on the high-pressure freezing of spatially constrained whole C. elegans hermaphrodites in a combination of cryoprotectants to identify embryonic cells in metaphase by in situ cryo-fluorescence microscopy. The screened worms were then freeze substituted, resin embedded and further prepared such that the targeted cells were successfully located and imaged by focused ion beam scanning electron microscopy (FIB-SEM). We reconstructed the targeted metaphase structure and also correlated an intriguing punctate fluorescence signal to a H2B-enriched putative polar body autophagosome in an adjacent cell undergoing telophase. By enabling cryo-fluorescence microscopy of thick samples, our workflow can thus be used to trap and image transient structures in C. elegans or similar organisms in a near-native state, and then reconstruct their corresponding cellular architectures at high resolution and in 3-D by correlative volume EM.

The many phases of anaphase.

Anaphase is the stage of the cell cycle in which duplicated chromosomes separate and move towards opposite poles of the cell. Although its chromosome movements have always been viewed as majestic, until recently anaphase lacked obvious landmarks of regulation. The picture has changed with numerous recent studies that have highlighted the raison d'être of anaphase. It is now known to be associated with a series of regulatory pathways that promote a switch from high to low cyclin-dependent kinase activity--an essential feature for proper mitotic exit. The balance between protein phosphorylation and protein dephosphorylation drives and coordinates diverse processes such as chromosome movement, spindle dynamics and cleavage furrow formation. This well-ordered sequence of events is central to successful mitosis.

Tracking Germline Stem Cell Dynamics In Vivo in C. elegans Using Photoconversion.

The maintenance of many adult tissues depends on stem cell systems, which must balance proliferation and differentiation. To understand the properties of adult stem cell systems, one powerful tool is visualization of the cell dynamics in vivo. Here we describe a protocol to track cells in the germline progenitor zone (which includes germline stem cells) in live C. elegans adult worms. Tracking is achieved by using a genetically encoded photoconvertible fluorescent protein, where photoconversion is used to mark cells of interest and their descendants. Individual worms are immobilized, the cells of interest are selected for photoconversion, and the worms are then recovered to plates and imaged at later timepoints. This allows longitudinal studies of individual worms, providing valuable information regarding germline stem cell dynamics.

C. elegans pronuclei fuse after fertilization through a novel membrane structure.

After fertilization, parental genomes are enclosed in two separate pronuclei. In Caenorhabditis elegans, and possibly other organisms, when the two pronuclei first meet, the parental genomes are separated by four pronuclear membranes. To understand how these membranes are breached to allow merging of parental genomes we used focused ion beam scanning electron microscopy (FIB-SEM) to study the architecture of the pronuclear membranes at nanometer-scale resolution. We find that at metaphase, the interface between the two pronuclei is composed of two membranes perforated by fenestrations ranging from tens of nanometers to several microns in diameter. The parental chromosomes come in contact through one of the large fenestrations. Surrounding this fenestrated, two-membrane region is a novel membrane structure, a three-way sheet junction, where the four membranes of the two pronuclei fuse and become two. In the plk-1 mutant, where parental genomes fail to merge, these junctions are absent, suggesting that three-way sheet junctions are needed for formation of a diploid genome.

Getting (chromosomes) loaded--a new role for timeless.

A recent study in C. elegans reveals an unanticipated link between sister chromatid cohesion and the TIM-1 protein, a homolog of the Drosophila circadian rhythm protein TIMELESS. The phenotypes of tim-1 mutants suggest that cohesin subunits load onto chromosomes in a stepwise manner. Whether TIM-1 is also involved in circadian rhythms is discussed.

A role for the FEAR pathway in nuclear positioning during anaphase.

In budding yeast, cells lacking separase function exit mitosis with an undivided nucleus localized to the daughter cell. Here we show that the inability to separate sister chromatids per se is not sufficient to cause the daughter preference. Rather, separase affects nuclear positioning as part of the Cdc14 early anaphase release (FEAR) pathway. The role of the FEAR pathway in nuclear positioning is exerted during anaphase and is not shared by the mitotic exit network. We find that the nuclear segregation defect in FEAR mutants does not stem from nonfunctional spindle poles or the absence of cytoplasmic microtubules. Instead, the concomitant inactivation of sister chromatid separation and the FEAR pathway uncovered a mother-directed force in anaphase that was previously masked by the elongating spindle. We propose that at anaphase onset, the FEAR pathway activates cytoplasmic microtubule-associated forces that facilitate chromosome segregation to the mother cell.

Yeast nuclear envelope subdomains with distinct abilities to resist membrane expansion.

Little is known about what dictates the round shape of the yeast Saccharomyces cerevisiae nucleus. In spo7Delta mutants, the nucleus is misshapen, exhibiting a single protrusion. The Spo7 protein is part of a phosphatase complex that represses phospholipid biosynthesis. Here, we report that the nuclear protrusion of spo7Delta mutants colocalizes with the nucleolus, whereas the nuclear compartment containing the bulk of the DNA is unaffected. Using strains in which the nucleolus is not intimately associated with the nuclear envelope, we show that the single nuclear protrusion of spo7Delta mutants is not a result of nucleolar expansion, but rather a property of the nuclear membrane. We found that in spo7Delta mutants the peripheral endoplasmic reticulum (ER) membrane was also expanded. Because the nuclear membrane and the ER are contiguous, this finding indicates that in spo7Delta mutants all ER membranes, with the exception of the membrane surrounding the bulk of the DNA, undergo expansion. Our results suggest that the nuclear envelope has distinct domains that differ in their ability to resist membrane expansion in response to increased phospholipid biosynthesis. We further propose that in budding yeast there is a mechanism, or structure, that restricts nuclear membrane expansion around the bulk of the DNA.

Chromosome cohesion: ring around the sisters?

Sister chromatid cohesion is a key aspect of accurate chromosome transmission during mitosis, yet little is known about the structure of cohesin, the protein complex that links the two sister chromatids. Recent studies shed light on the structure of the cohesin complex, leading to intriguing models that could explain how sister chromatids are held together.

The Multiple Ways Nuclei Scale on a Multinucleated Muscle Cell Scale.

In mononucleated cells, nuclear size scales with cell size, but does this relationship extend to multinucleated cells? In this issue of Developmental Cell,Windner et al. (2019) examine scaling of nuclei in multinucleated Drosophila muscle fibers and identify global and local cellular inputs that contribute to nuclear size regulation.