Automated profiling of gene function during embryonic development.

Systematic functional profiling of the gene set that directs embryonic development is an important challenge. To tackle this challenge, we used 4D imaging of C. elegans embryogenesis to capture the effects of 500 gene knockdowns and developed an automated approach to compare developmental phenotypes. The automated approach quantifies features-including germ layer cell numbers, tissue position, and tissue shape-to generate temporal curves whose parameterization yields numerical phenotypic signatures. In conjunction with a new similarity metric that operates across phenotypic space, these signatures enabled the generation of ranked lists of genes predicted to have similar functions, accessible in the PhenoBank web portal, for ∼25% of essential development genes. The approach identified new gene and pathway relationships in cell fate specification and morphogenesis and highlighted the utilization of specialized energy generation pathways during embryogenesis. Collectively, the effort establishes the foundation for comprehensive analysis of the gene set that builds a multicellular organism.

Evolutionary Dynamics and Molecular Mechanisms of HORMA Domain Protein Signaling.

Controlled assembly and disassembly of multi-protein complexes is central to cellular signaling. Proteins of the widespread and functionally diverse HORMA family nucleate assembly of signaling complexes by binding short peptide motifs through a distinctive safety-belt mechanism. HORMA proteins are now understood as key signaling proteins across kingdoms, serving as infection sensors in a bacterial immune system and playing central roles in eukaryotic cell cycle, genome stability, sexual reproduction, and cellular homeostasis pathways. Here, we describe how HORMA proteins' unique ability to adopt multiple conformational states underlies their functions in these diverse contexts. We also outline how a dedicated AAA+ ATPase regulator, Pch2/TRIP13, manipulates HORMA proteins' conformational states to activate or inactivate signaling in different cellular contexts. The emergence of Pch2/TRIP13 as a lynchpin for HORMA protein action in multiple genome-maintenance pathways accounts for its frequent misregulation in human cancers and highlights TRIP13 as a novel therapeutic target.

A TOGgle for Tension at Kinetochores.

Differential stability of kinetochore-microtubule attachments at low versus high tension is critical for accurate chromosome segregation. Miller et al. find that a TOG domain microtubule-binding protein imparts intrinsic tension selectivity to kinetochore-microtubule attachments.

A Small RNA-Catalytic Argonaute Pathway Tunes Germline Transcript Levels to Ensure Embryonic Divisions.

Multiple division cycles without growth are a characteristic feature of early embryogenesis. The female germline loads proteins and RNAs into oocytes to support these divisions, which lack many quality control mechanisms operating in somatic cells undergoing growth. Here, we describe a small RNA-Argonaute pathway that ensures early embryonic divisions in C. elegans by employing catalytic slicing activity to broadly tune, instead of silence, germline gene expression. Misregulation of one target, a kinesin-13 microtubule depolymerase, underlies a major phenotype associated with pathway loss. Tuning of target transcript levels is guided by the density of homologous small RNAs, whose generation must ultimately be related to target sequence. Thus, the tuning action of a small RNA-catalytic Argonaute pathway generates oocytes capable of supporting embryogenesis. We speculate that the specialized nature of germline chromatin led to the emergence of small RNA-catalytic Argonaute pathways in the female germline as a post-transcriptional control layer to optimize oocyte composition.

APC7 mediates ubiquitin signaling in constitutive heterochromatin in the developing mammalian brain.

Neurodevelopmental cognitive disorders provide insights into mechanisms of human brain development. Here, we report an intellectual disability syndrome caused by the loss of APC7, a core component of the E3 ubiquitin ligase anaphase promoting complex (APC). In mechanistic studies, we uncover a critical role for APC7 during the recruitment and ubiquitination of APC substrates. In proteomics analyses of the brain from mice harboring the patient-specific APC7 mutation, we identify the chromatin-associated protein Ki-67 as an APC7-dependent substrate of the APC in neurons. Conditional knockout of the APC coactivator protein Cdh1, but not Cdc20, leads to the accumulation of Ki-67 protein in neurons in vivo, suggesting that APC7 is required for the function of Cdh1-APC in the brain. Deregulated neuronal Ki-67 upon APC7 loss localizes predominantly to constitutive heterochromatin. Our findings define an essential function for APC7 and Cdh1-APC in neuronal heterochromatin regulation, with implications for understanding human brain development and disease.

Visualization of eukaryotic DNA mismatch repair reveals distinct recognition and repair intermediates.

DNA mismatch repair (MMR) increases replication fidelity by eliminating mispaired bases resulting from replication errors. In Saccharomyces cerevisiae, mispairs are primarily detected by the Msh2-Msh6 complex and corrected following recruitment of the Mlh1-Pms1 complex. Here, we visualized functional fluorescent versions of Msh2-Msh6 and Mlh1-Pms1 in living cells. We found that the Msh2-Msh6 complex is an S phase component of replication centers independent of mispaired bases; this localized pool accounted for 10%-15% of MMR in wild-type cells but was essential for MMR in the absence of Exo1. Unexpectedly, Mlh1-Pms1 formed nuclear foci that, although dependent on Msh2-Msh6 for formation, rarely colocalized with Msh2-Msh6 replication-associated foci. Mlh1-Pms1 foci increased when the number of mispaired bases was increased; in contrast, Msh2-Msh6 foci were unaffected. These findings suggest the presence of replication machinery-coupled and -independent pathways for mispair recognition by Msh2-Msh6, which direct formation of superstoichiometric Mlh1-Pms1 foci that represent sites of active MMR.

A high-resolution C. elegans essential gene network based on phenotypic profiling of a complex tissue.

High-content screening for gene profiling has generally been limited to single cells. Here, we explore an alternative approach-profiling gene function by analyzing effects of gene knockdowns on the architecture of a complex tissue in a multicellular organism. We profile 554 essential C. elegans genes by imaging gonad architecture and scoring 94 phenotypic features. To generate a reference for evaluating methods for network construction, genes were manually partitioned into 102 phenotypic classes, predicting functions for uncharacterized genes across diverse cellular processes. Using this classification as a benchmark, we developed a robust computational method for constructing gene networks from high-content profiles based on a network context-dependent measure that ranks the significance of links between genes. Our analysis reveals that multi-parametric profiling in a complex tissue yields functional maps with a resolution similar to genetic interaction-based profiling in unicellular eukaryotes-pinpointing subunits of macromolecular complexes and components functioning in common cellular processes.

TRIM37 controls cancer-specific vulnerability to PLK4 inhibition.

Centrosomes catalyse the formation of microtubules needed to assemble the mitotic spindle apparatus1. Centrosomes themselves duplicate once per cell cycle, in a process that is controlled by the serine/threonine protein kinase PLK4 (refs. 2,3). When PLK4 is chemically inhibited, cell division proceeds without centrosome duplication, generating centrosome-less cells that exhibit delayed, acentrosomal spindle assembly4. Whether PLK4 inhibitors can be leveraged as a treatment for cancer is not yet clear. Here we show that acentrosomal spindle assembly following PLK4 inhibition depends on levels of the centrosomal ubiquitin ligase TRIM37. Low TRIM37 levels accelerate acentrosomal spindle assembly and improve proliferation following PLK4 inhibition, whereas high TRIM37 levels inhibit acentrosomal spindle assembly, leading to mitotic failure and cessation of proliferation. The Chr17q region containing the TRIM37 gene is frequently amplified in neuroblastoma and in breast cancer5-8, rendering these cancer types highly sensitive to PLK4 inhibition. We find that inactivating TRIM37 improves acentrosomal mitosis because TRIM37 prevents PLK4 from self-assembling into centrosome-independent condensates that serve as ectopic microtubule-organizing centres. By contrast, elevated TRIM37 expression inhibits acentrosomal spindle assembly through a distinct mechanism that involves degradation of the centrosomal component CEP192. Thus, TRIM37 is an essential determinant of mitotic vulnerability to PLK4 inhibition. Linkage of TRIM37 to prevalent cancer-associated genomic changes-including 17q gain in neuroblastoma and 17q23 amplification in breast cancer-may offer an opportunity to use PLK4 inhibition to trigger selective mitotic failure and provide new avenues to treatments for these cancers.

Kinetochores accelerate or delay APC/C activation by directing Cdc20 to opposing fates.

Mitotic duration is determined by activation of the anaphase-promoting complex/cyclosome (APC/C) bound to its coactivator, Cdc20. Kinetochores, the microtubule-interacting machines on chromosomes, restrain mitotic exit when not attached to spindle microtubules by generating a Cdc20-containing complex that inhibits the APC/C. Here, we show that flux of Cdc20 through kinetochores also accelerates mitotic exit by promoting its dephosphorylation by kinetochore-localized protein phosphatase 1, which allows Cdc20 to activate the APC/C. Both APC/C activation and inhibition depend on Cdc20 fluxing through the same binding site at kinetochores. The microtubule attachment status of kinetochores therefore optimizes mitotic duration by controlling the balance between opposing Cdc20 fates.

Structural memory in the contractile ring makes the duration of cytokinesis independent of cell size.

Cytokinesis is accomplished by constriction of a cortical contractile ring. We show that during the early embryonic divisions in C. elegans, ring constriction occurs in two phases--an initial phase at a constant rate followed by a second phase during which the constriction rate decreases in proportion to ring perimeter. Cytokinesis completes in the same amount of time, despite the reduction in cell size during successive divisions, due to a strict proportionality between initial ring size and the constant constriction rate. During closure, the myosin motor in the ring decreases in proportion to perimeter without turning over. We propose a "contractile unit" model to explain how the ring retains a structural memory of its initial size as it disassembles. The scalability of constriction may facilitate coordination of mitotic events and cytokinesis when cell size, and hence the distance traversed by the ring, varies during embryogenesis and in other contexts.

Protein architecture of the human kinetochore microtubule attachment site.

Chromosome segregation requires assembly of kinetochores on centromeric chromatin to mediate interactions with spindle microtubules and control cell-cycle progression. To elucidate the protein architecture of human kinetochores, we developed a two-color fluorescence light microscopy method that measures average label separation, Delta, at <5 nm accuracy. Delta analysis of 16 proteins representing core structural complexes spanning the centromeric chromatin-microtubule interface, when correlated with mechanical states of spindle-attached kinetochores, provided a nanometer-scale map of protein position and mechanical properties of protein linkages. Treatment with taxol, which suppresses microtubule dynamics and activates the spindle checkpoint, revealed a specific switch in kinetochore architecture. Cumulatively, Delta analysis revealed that compliant linkages are restricted to the proximity of chromatin, suggested a model for how the KMN (KNL1/Mis12 complex/Ndc80 complex) network provides microtubule attachment and generates pulling forces from depolymerization, and identified an intrakinetochore molecular switch that may function in controlling checkpoint activity.

Spindle assembly checkpoint activation and silencing at kinetochores.

The spindle assembly checkpoint (SAC) is a surveillance mechanism that promotes accurate chromosome segregation in mitosis. The checkpoint senses the attachment state of kinetochores, the proteinaceous structures that assemble onto chromosomes in mitosis in order to mediate their interaction with spindle microtubules. When unattached, kinetochores generate a diffusible inhibitor that blocks the activity of the anaphase-promoting complex/cyclosome (APC/C), an E3 ubiquitin ligase required for sister chromatid separation and exit from mitosis. Work from the past decade has greatly illuminated our understanding of the mechanisms by which the diffusible inhibitor is assembled and how it inhibits the APC/C. However, less is understood about how SAC proteins are recruited to kinetochores in the absence of microtubule attachment, how the kinetochore catalyzes formation of the diffusible inhibitor, and how attachments silence the SAC at the kinetochore. Here, we summarize current understanding of the mechanisms that activate and silence the SAC at kinetochores and highlight open questions for future investigation.

Fibrils connect microtubule tips with kinetochores: a mechanism to couple tubulin dynamics to chromosome motion.

Kinetochores of mitotic chromosomes are coupled to spindle microtubules in ways that allow the energy from tubulin dynamics to drive chromosome motion. Most kinetochore-associated microtubule ends display curving "protofilaments," strands of tubulin dimers that bend away from the microtubule axis. Both a kinetochore "plate" and an encircling, ring-shaped protein complex have been proposed to link protofilament bending to poleward chromosome motion. Here we show by electron tomography that slender fibrils connect curved protofilaments directly to the inner kinetochore. Fibril-protofilament associations correlate with a local straightening of the flared protofilaments. Theoretical analysis reveals that protofilament-fibril connections would be efficient couplers for chromosome motion, and experimental work on two very different kinetochore components suggests that filamentous proteins can couple shortening microtubules to cargo movements. These analyses define a ring-independent mechanism for harnessing microtubule dynamics directly to chromosome movement.

A high-content imaging approach to profile C. elegans embryonic development.

The Caenorhabditis elegans embryo is an important model for analyzing mechanisms of cell fate specification and tissue morphogenesis. Sophisticated lineage-tracing approaches for analyzing embryogenesis have been developed but are labor intensive and do not naturally integrate morphogenetic readouts. To enable the rapid classification of developmental phenotypes, we developed a high-content method that employs two custom strains: a Germ Layer strain that expresses nuclear markers in the ectoderm, mesoderm and endoderm/pharynx; and a Morphogenesis strain that expresses markers labeling epidermal cell junctions and the neuronal cell surface. We describe a procedure that allows simultaneous live imaging of development in 80-100 embryos and provide a custom program that generates cropped, oriented image stacks of individual embryos to facilitate analysis. We demonstrate the utility of our method by perturbing 40 previously characterized developmental genes in variants of the two strains containing RNAi-sensitizing mutations. The resulting datasets yielded distinct, reproducible signature phenotypes for a broad spectrum of genes that are involved in cell fate specification and morphogenesis. In addition, our analysis provides new in vivo evidence for MBK-2 function in mesoderm fate specification and LET-381 function in elongation.

PCNA and Msh2-Msh6 activate an Mlh1-Pms1 endonuclease pathway required for Exo1-independent mismatch repair.

Genetic evidence has implicated multiple pathways in eukaryotic DNA mismatch repair (MMR) downstream of mispair recognition and Mlh1-Pms1 recruitment, including Exonuclease 1 (Exo1)-dependent and -independent pathways. We identified 14 mutations in POL30, which encodes PCNA in Saccharomyces cerevisiae, specific to Exo1-independent MMR. The mutations identified affected amino acids at three distinct sites on the PCNA structure. Multiple mutant PCNA proteins had defects either in trimerization and Msh2-Msh6 binding or in activation of the Mlh1-Pms1 endonuclease that initiates excision during MMR. The latter class of mutations led to hyperaccumulation of repair intermediate Mlh1-Pms1 foci and were enhanced by an msh6 mutation that disrupted the Msh2-Msh6 interaction with PCNA. These results reveal a central role for PCNA in the Exo1-independent MMR pathway and suggest that Msh2-Msh6 localizes PCNA to repair sites after mispair recognition to activate the Mlh1-Pms1 endonuclease for initiating Exo1-dependent repair or for driving progressive excision in Exo1-independent repair.

Spatial control of phospholipid flux restricts endoplasmic reticulum sheet formation to allow nuclear envelope breakdown.

The nuclear envelope is a subdomain of the endoplasmic reticulum (ER). Here we characterize CNEP-1 (CTD [C-terminal domain] nuclear envelope phosphatase-1), a nuclear envelope-enriched activator of the ER-associated phosphatidic acid phosphatase lipin that promotes synthesis of major membrane phospholipids over phosphatidylinositol (PI). CNEP-1 inhibition led to ectopic ER sheets in the vicinity of the nucleus that encased the nuclear envelope and interfered with nuclear envelope breakdown (NEBD) during cell division. Reducing PI synthesis suppressed these phenotypes, indicating that CNEP-1 spatially regulates phospholipid flux, biasing it away from PI production in the vicinity of the nuclear envelope to prevent excess ER sheet formation and NEBD defects.

Pharmacological convergence reveals a lipid pathway that regulates C. elegans lifespan.

Phenotypic screening has identified small-molecule modulators of aging, but the mechanism of compound action often remains opaque due to the complexities of mapping protein targets in whole organisms. Here, we combine a library of covalent inhibitors with activity-based protein profiling to coordinately discover bioactive compounds and protein targets that extend lifespan in Caenorhabditis elegans. We identify JZL184-an inhibitor of the mammalian endocannabinoid (eCB) hydrolase monoacylglycerol lipase (MAGL or MGLL)-as a potent inducer of longevity, a result that was initially perplexing as C. elegans does not possess an MAGL ortholog. We instead identify FAAH-4 as a principal target of JZL184 and show that this enzyme, despite lacking homology with MAGL, performs the equivalent metabolic function of degrading eCB-related monoacylglycerides in C. elegans. Small-molecule phenotypic screening thus illuminates pure pharmacological connections marking convergent metabolic functions in distantly related organisms, implicating the FAAH-4/monoacylglyceride pathway as a regulator of lifespan in C. elegans.

Cdc73 suppresses genome instability by mediating telomere homeostasis.

Defects in the genes encoding the Paf1 complex can cause increased genome instability. Loss of Paf1, Cdc73, and Ctr9, but not Rtf1 or Leo1, caused increased accumulation of gross chromosomal rearrangements (GCRs). Combining the cdc73Δ mutation with individual deletions of 43 other genes, including TEL1 and YKU80, which are involved in telomere maintenance, resulted in synergistic increases in GCR rates. Whole genome sequence analysis of GCRs indicated that there were reduced relative rates of GCRs mediated by de novo telomere additions and increased rates of translocations and inverted duplications in cdc73Δ single and double mutants. Analysis of telomere lengths and telomeric gene silencing in strains containing different combinations of cdc73Δ, tel1Δ and yku80Δ mutations suggested that combinations of these mutations caused increased defects in telomere maintenance. A deletion analysis of Cdc73 revealed that a central 105 amino acid region was necessary and sufficient for suppressing the defects observed in cdc73Δ strains; this region was required for the binding of Cdc73 to the Paf1 complex through Ctr9 and for nuclear localization of Cdc73. Taken together, these data suggest that the increased GCR rate of cdc73Δ single and double mutants is due to partial telomere dysfunction and that Ctr9 and Paf1 play a central role in the Paf1 complex potentially by scaffolding the Paf1 complex subunits or by mediating recruitment of the Paf1 complex to the different processes it functions in.

A toolkit for GFP-mediated tissue-specific protein degradation in C. elegans.

Proteins that are essential for embryo production, cell division and early embryonic events are frequently reused later in embryogenesis, during organismal development or in the adult. Examining protein function across these different biological contexts requires tissue-specific perturbation. Here, we describe a method that uses expression of a fusion between a GFP-targeting nanobody and a SOCS-box containing ubiquitin ligase adaptor to target GFP-tagged proteins for degradation. When combined with endogenous locus GFP tagging by CRISPR-Cas9 or with rescue of a null mutant with a GFP fusion, this approach enables routine and efficient tissue-specific protein ablation. We show that this approach works in multiple tissues - the epidermis, intestine, body wall muscle, ciliated sensory neurons and touch receptor neurons - where it recapitulates expected loss-of-function mutant phenotypes. The transgene toolkit and the strain set described here will complement existing approaches to enable routine analysis of the tissue-specific roles of C. elegans proteins.

Molecular architecture of the kinetochore-microtubule interface.

Segregation of the replicated genome during cell division in eukaryotes requires the kinetochore to link centromeric DNA to spindle microtubules. The kinetochore is composed of a number of conserved protein complexes that direct its specification and assembly, bind to spindle microtubules and regulate chromosome segregation. Recent studies have identified more than 80 kinetochore components, and are revealing how these proteins are organized into the higher order kinetochore structure, as well as how they function to achieve proper chromosome segregation.