Tracking and Quantifying Developmental Processes in <em>C. elegans</em> Using Open-source Tools

Quantitatively capturing developmental processes is crucial to derive mechanistic models and key to identify and describe mutant phenotypes. Here ...

Goethe University

Apoptotic cell death and survival is controlled by pro- and antiapoptotic proteins. Because these proteins act on each other, cell fate is dictated by the relative activity of pro- versus antiapoptotic proteins. Here we report that BRUCE, a conserved 528 kDa peripheral membrane protein of the trans-Golgi network, protects cells against apoptosis and functions as an inhibitor of apoptosis (IAP). By using wild-type and mutant forms we show that BRUCE inhibits caspase activity and apoptosis depending on its BIR domain. Upon apoptosis induction, BRUCE is antagonized by three mechanisms: first, through binding to Smac; second, by the protease HtrA2; and third, by caspase-mediated cleavage. In addition to its IAP activity BRUCE has the distinctive property of functioning as a chimeric E2/E3 ubiquitin ligase with Smac being a substrate. Our work suggests that, owing to its two activities and its localization, BRUCE may function as a specialized regulator of cell death pathways.

Dual role of BRUCE as an antiapoptotic IAP and a chimeric E2/E3 ubiquitin ligase.

Cytokinesis involves the formation of a cleavage furrow, followed by abscission, the cutting of the midbody channel, the final bridge between dividing cells. Recently, the midbody ring became known as central for abscission, but its regulation remains enigmatic. Here, we identify BRUCE, a 528 kDa multifunctional protein, which processes ubiquitin-conjugating activity, as a major regulator of abscission. During cytokinesis, BRUCE moves from the vesicular system to the midbody ring and serves as a platform for the membrane delivery machinery and mitotic regulators. Depletion of BRUCE in cell cultures causes defective abscission and cytokinesis-associated apoptosis, accompanied by a block of vesicular targeting and defective formation of the midbody and the midbody ring. Notably, ubiquitin relocalizes from midbody microtubules to the midbody ring during cytokinesis, and depletion of BRUCE disrupts this process. We propose that BRUCE coordinates multiple steps required for abscission and that ubiquitylation may be a crucial trigger.

Final stages of cytokinesis and midbody ring formation are controlled by BRUCE.

At the end of cytokinesis, the dividing cells are connected by an intercellular bridge, containing the midbody along with a single, densely ubiquitylated, circular structure called the midbody ring (MR). Recent studies revealed that the MR serves as a target site for membrane delivery and as a physical barrier between the prospective daughter cells. The MR materializes in telophase, localizes to the intercellular bridge during cytokinesis, and moves asymmetrically into one cell after abscission. Daughter cells rarely accumulate MRs of previous divisions, but how these large structures finally disappear remains unknown. Here, we show that MRs are discarded by autophagy, which involves their sequestration into autophagosomes and delivery to lysosomes for degradation. Notably, autophagy factors, such as the ubiquitin adaptor p62 (Refs 4, 5) and the ubiquitin-related protein Atg8 (ref. 6), associate with the MR during abscission, suggesting that autophagy is coupled to cytokinesis. Moreover, MRs accumulate in cells of patients with lysosomal storage disorders, indicating that defective MR disposal is characteristic of these diseases. Thus our findings suggest that autophagy has a broader role than previously assumed, and that cell renovation by clearing from superfluous large macromolecular assemblies, such as MRs, is an important autophagic function.

Midbody ring disposal by autophagy is a post-abscission event of cytokinesis.

After a cell has segregated sister chromatids in mitosis, the central part of the mitotic spindle differentiates into a specialized organelle, the midbody, lying in the center of the intercellular bridge that connects the two daughter cells. In the concluding step of cytokinesis, membrane insertion seals the cytoplasmic channel resulting in the physical separation of the daughters. A circular protein-dense structure at the midbody's focus, called Flemming body or midbody ring (MR), is considered to be the integrating platform for the following: The accumulation and remodeling of membranes, the ubiquitination of several of its constituents, the segregation of signaling components, and the sequestration of fate-determining factors. We recently demonstrated that MRs, after they become ubiquitinated, are found inside autophagosomes and finally deteriorate when these autophagosomes mature into lysosomes. Strikingly, p62/SQSTM1, an adaptor protein that delivers ubiquitinated proteins for autophagosomal degradation, is recruited to the MR at the time of its ubiquitination and is required for its efficient degradation. Furthermore, downregulating autophagy or lysosomal degradation showed marked accumulation of MRs, suggesting that clearing of MRs by autophagy represents a process directly coupled to cytokinesis. Considering our own recent findings and several reports from other labs, we suggest a sequential model of late stages of cytokinesis that includes (a) the formation of a binding surface by ubiquitination of MR components, (b) recruitment of ESCRT complexes for membrane remodeling and (c) handover of the membrane-enveloped MR either to autophagosomal degradation or exocytosis.

Dual control of cytokinesis by the ubiquitin and autophagy pathways.

Left-right (LR) patterning is an intriguing but poorly understood process of bilaterian embryogenesis. We report a mechanism for LR patterning in C. elegans in which the embryo uncouples its midline from the anteroposterior (AP) axis. Specifically, the eight-cell embryo establishes a midline that is tilted rightward from the AP axis and positions more cells on the left, allowing subsequent differential LR fate inductions. To establish the tilted midline, cells exhibit LR asymmetric protrusions and a handed collective movement. This process, termed chiral morphogenesis, involves differential regulation of cortical contractility between a pair of sister cells that are bilateral counterparts fate-wise and is activated by noncanonical Wnt signaling. Chiral morphogenesis is timed by the cytokinetic furrow of a neighbor of the sister pair, providing a developmental clock and an unexpected signaling interaction between the contractile ring and the adjacent cells.

Chiral forces organize left-right patterning in C. elegans by uncoupling midline and anteroposterior axis.

The development of bilateral symmetry during the evolution of species probably 600 million years ago brought about several important innovations: It fostered efficient locomotion, streamlining and favored the development of a central nervous system through cephalization. However, to increase their functional capacities, many organisms exhibit chirality by breaking their superficial left-right (l-r) symmetry, which manifests in the lateralization of the nervous system or the l-r asymmetry of internal organs. In most bilateria, the mechanisms that maintain consistent l-r asymmetry throughout development are poorly understood. This review highlights insights into mechanisms that couple early embryonic l-r symmetry breaking to subsequent l-r patterning in the roundworm Caenorhabditis elegans. A recently identified strategy for l-r patterning in the early C. elegans embryo is discussed, the spatial separation of midline and anteroposterior axis, which relies on a rotational cellular rearrangement and non-canonical Wnt signaling. Evidence for a general relevance of rotational/torsional rearrangements during organismal l-r patterning and for non-canonical Wnt signaling/planar cell polarity as a common signaling mechanism to maintain l-r asymmetry is presented.

Left-right patterning in the C. elegans embryo: Unique mechanisms and common principles.

Fighting mycobacteria through ISGylation.

Gastrulation is a key transition in embryogenesis; it requires self-organized cellular coordination, which has to be both robust to allow efficient development and plastic to provide adaptability. Despite the conservation of gastrulation as a key event in Metazoan embryogenesis, the morphogenetic mechanisms of self-organization (how global order or coordination can arise from local interactions) are poorly understood.

Actomyosin-based self-organization of cell internalization during C. elegans gastrulation.

Pulmonary aspergilloma: a treatment challenge in sub-Saharan Africa.

The Team Science Toolkit: enhancing research collaboration through online knowledge sharing.

Cortical flows mediate anteroposterior polarization in Caenorhabditis elegans by generating two mutually exclusive membrane domains. However, factors downstream of anteroposterior polarity that establish the dorsoventral axis remain elusive. Here, we show that rotational cortical flow orthogonal to the anteroposterior axis during the division of the AB blastomere in the two-cell embryo positions the cytokinetic midbody remnant of the previous division asymmetrically at the future ventral side of the embryo. In the neighboring P1 blastomere, astral microtubules contact a transient PAR-2-dependent actin coat that forms asymmetrically onto the midbody remnant-P1 interface. Ablation of the midbody remnant or perturbation of rotational cortical flow reveals that microtubule-midbody remnant contacts are crucial for P1 spindle rotation and dorsoventral axis formation. Thus, our findings suggest a mechanism for dorsoventral patterning that relies on coupling of anteroposterior polarity, rotational cortical flow, midbody remnant positioning, and spindle orientation.

Coupling of rotational cortical flow, asymmetric midbody positioning, and spindle rotation mediates dorsoventral axis formation in C. elegans.

Asymmetric cell divisions combine cell division with fate specification and one general model of how this is achieved was proposed already decades ago(1,2): During interphase, the cell polarity axis is specified, followed by orientation of the spindle along the polarity axis and segregation of fate determinants along the polarity axis during mitosis. In most cells, the polarity axis and the spindle will usually align with the long axis that the cell had before division, also called Hertwig's rule(3-6). In the C. elegans embryo, the first polarity axis also forms along the long axis of the embryo by enrichment of myosin in the anterior(7) and formation of mutually exclusive anterior and posterior cortical polarity domains, mediated through directional cortical contractile flow(8-10). The directionality of this flow is determined by an extrinsic cue, the entry of the sperm, which inhibits Rho-dependent myosin activation at the future posterior pole by bringing with it the Rho GTPase activating protein CYK-4(11,12). Moreover, since there is no previous division 'history' before the first cleavage, mechanisms have to ensure that the nucleus-centrosome complex undergoes a 90 degree rotation so that the spindle can subsequently elongate along the long axis(13-15). Additional mechanisms ensure that the site of cleavage is perpendicular to the long axis(16,17). Hence, tight coupling of an extrinsic cue to intrinsic polarity formation and spindle elongation enables alignment of the division orientation with the long axis of the organism and successful segregation of fate determinants.

A function for the midbody remnant in embryonic patterning.

Novel tuberculosis vaccines should be safe, immunogenic, and effective in various population groups, including HIV-infected individuals. In this phase II multi-centre, double-blind, placebo-controlled trial, the safety and immunogenicity of the novel H1/IC31 vaccine, a fusion protein of Ag85B-ESAT-6 (H1) formulated with the adjuvant IC31, was evaluated in HIV-infected adults.

Safety and immunogenicity of H1/IC31®, an adjuvanted TB subunit vaccine, in HIV-infected adults with CD4+ lymphocyte counts greater than 350 cells/mm3: a phase II, multi-centre, double-blind, randomized, placebo-controlled trial.

Christian Pohl

Buchmann Institute for Molecular Life Sciences and Institute of Biochemistry II,
School of Medicine,
Buchmann Institute for Molecular Life Sciences and Institute of Biochemistry II, School of Medicine

Goethe University

Tracking and Quantifying Developmental Processes in C. elegans Using Open-source Tools

Christian Pohl

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Goethe University

Tracking and Quantifying Developmental Processes in C. elegans Using Open-source Tools

Buchmann Institute for Molecular Life Sciences and Institute of Biochemistry II,
School of Medicine,
Buchmann Institute for Molecular Life Sciences and Institute of Biochemistry II, School of Medicine