Molecular Biology of the Cell

Cortical positioning of oocyte meiotic spindles is required for reducing chromosome number by segregating chromosomes into polar bodies. In C. elegans, kinesin-1 transports membrane-bound organelles inward into a central ball that is hypothesized to push the meiotic spindle outward. Using focused ion beam scanning electron microscopy, we found that the percent volume occupied by membrane-bound organelles was 28% in a mature oocyte that has completed endocytosis of the yolk precursor, vitellogenin, compared to 17% in a less mature oocyte that has not yet endocytosed vitellogenin. We therefore hypothesized that blocking endocytosis of vitellogenin would reduce the size of the central ball of organelles packed inward by kinesin-1 and limit outward movement of the spindle. Depletion of vitellogenin or proteins required for endocytosis resulted in a smaller central volume of kinesin-coated organelles and defective spindle positioning. A computer simulation of a simple exclusion model faithfully recapitulated wildtype and mutant spindle position phenotypes. These results support a volume exclusion model for positioning acentriolar meiotic spindles.

The opportunistic human pathogen Candida albicans depends on the unfolded protein response (UPR) for cell wall integrity, antifungal tolerance, filamentous growth, and virulence. The UPR is driven by the conserved transmembrane sensor Ire1, which is activated either by misfolded proteins through its luminal domain or by lipid bilayer stress (LBS) through its transmembrane domain. In budding yeast, these two activation modes deploy divergent transcriptional programs. Whether the requirement for these two input domains is separable in C. albicans, where the cell membrane and cell wall are themselves the targets of major antifungal drug classes, remains unknown. Here, we engineered a C. albicans strain expressing Ire1 lacking an intact luminal domain (ire1{Delta}LD), which no longer detects proteotoxic stress. The ire1{Delta}LD strain grew in the presence of the azole antifungals fluconazole and miconazole but was highly sensitive to heat shock, cell wall stress, and the echinocandin caspofungin. It was also unable to sustain filamentous growth and showed reduced virulence in a Caenorhabditis elegans infection model. RNA sequencing revealed only modest changes to the steady-state transcriptome of ire1{Delta}LD cells. Together, these findings define a differential requirement for the input domains of C. albicans Ire1, uncoupling growth under azole-induced membrane stress from the cell wall, thermal, and virulence-associated outputs that depend on proteotoxic sensing, a distinction that could inform antifungal strategies targeting the UPR.

In the C. elegans reproductive system, oocyte transit through the spermatheca relies on parallel calcium and Rho-dependent signaling pathways to regulate actomyosin contractility. Here, we identify the conserved single-PDZ domain protein TXBP-3/TAX1BP3 as a dosage-dependent regulator of spermathecal contractility. TXBP-3 is expressed in contractile tissues, including the spermatheca and spermathecal-uterine (sp-ut) valve. Animals lacking txbp-3 do not display overt phenotypes or defects in ovulation. However, in nematodes expressing the genetically encoded calcium sensor GCaMP, deletion of txbp-3 results in more rapid transit of embryos through the spermatheca. Conversely, tissue-specific overexpression of TXBP-3 results in slower transit times due to hypercontraction of the spermathecal neck and sp-ut valve, causing oocyte severing and failures of embryos to exit the spermatheca. We find TXBP-3 promotes the active phosphorylated state of the calcium/calmodulin-dependent protein kinase II (CaMKII) UNC-43. When TXBP-3 is overexpressed or unc-43 is depleted, ovulations in which the embryo fails to exit show disrupted calcium signaling. Additionally, TXBP-3 functions independently of the RhoGEF RHGF-1 and in parallel with the RhoGAP SPV-1 to maintain the alignment of actin fiber bundles and protect embryo structural integrity. Together, our results establish TXBP-3 as a regulator of contractility, kinase activation, and cytoskeletal architecture in contractile tissues.

During cytokinesis in amphibian and echinoderm embryos, propagating waves of Rho activity and F-actin assembly - cortical excitability - are amplified and focused at the equatorial cortex by the mitotic spindle. Such cortical waves are proposed to result from fast positive feedback based on Rho and the Rho GEF, Ect2, and delayed negative feedback involving F-actin and the Rho GAP, RGA-3/4. Currently, this model is tenuous, in part because the relationship between F-actin and RGA-3/4 is not well understood. Therefore, we investigated how RGA-3/4 regulates F-actin structure both in vivo and in vitro. Full-length RGA-3/4, when expressed alone in Xenopus oocytes, localizes to and bundles cortical F-actin. In vitro, purified full-length RGA-3/4 protein directly binds to and bundles F-actin. TIRF microscopy revealed that RGA-3/4 binds to F-actin cooperatively and accumulates on the filaments before filament crosslinking events. Structure-function analyses demonstrated both the amino-terminal region and the intrinsically disordered region at the carboxyl-terminal portion of RGA-3/4 are essential for normal F-actin interaction. These results provide the first demonstration that RGA-3/4 is a direct, cooperative F-actin binding and bundling protein. We propose that its special features make RGA-3/4 uniquely suited to provide F-actin-dependent negative feedback during cortical excitability and cytokinesis.

The actomyosin cytoskeleton plays important roles in cell-cell adhesion by generating and responding to forces. Actin-binding proteins support actomyosin networks by reinforcing actin filaments, promoting actin remodeling, and transmitting forces to transmembrane adhesion proteins. One family of actin-binding proteins, LIM domain-containing proteins, is recruited to strained actin filaments. Here, we investigate two members of the LIM domain family, Zyxin and Lipoma-Preferred Partner (LPP), using the embryonic epithelium of Xenopus laevis, where actin-associated cell-cell junctions connect cells to promote tissue integrity and barrier function. Specifically, we compare Zyxin and LPP's response to increased tension at tricellular junctions (TCJs), sites of heightened mechanical strain within the tissue. Upon increased tension, Zyxin and LPP mechanoaccumulate to different extents relative to F-actin, suggesting distinct mechanisms. Our results demonstrate that Zyxin's and LPP's LIM domain-containing regions (LCRs) are sufficient for mechanoaccumulation as well as responsible for the difference in mechanoaccumulation, while their N-termini play a regulatory role in their mechanosensitive responses. Docking simulations show that a hydrophobic interaction between the LCR and the "crack site" that forms in F-actin filaments under high force may underlie the LCR's sensing of strained F-actin. Additionally, the docking simulations reveal that previously-identified conserved residues are present at the binding interface. Mutations of conserved residues in Zyxin or LPP reduce the extent of mechanoaccumulation at TCJs, supporting the docking prediction. Together, this study advances our understanding of Zyxin and LPP's sensing of strained actin at TCJs under mechanical challenge.

Inflammatory activation remodels the Golgi apparatus to meet increased secretory demand, yet whether these structural changes carry computationally accessible information about immune cell state has not been explored. Here we show that machine learning applied to imaging flow cytometry images of the Golgi can distinguish lipopolysaccharide (LPS) treated from control cells. We trained models on Golgi images from human THP-1 monocytes before and 16 h after LPS stimulation in vitro, and from circulating myeloid cells of mice 24 h after intratracheal LPS or saline in vivo. In each dataset, the best-performing model was a deep learning model trained directly on images rather than a classifier using predefined morphometric features. Cross-domain performance varied with the model, transfer direction and use of labelled target data: several deep learning models trained on mouse images classified THP-1 cells above chance without changing model parameters. Grad-CAM highlighted regions within or adjacent to the GM130 fluorescence pattern. These findings indicate that GM130 images carry information associated with LPS treatment that supports classification within and across the two datasets. To our knowledge, this is the first application of supervised deep learning to imaging flow cytometry images of a subcellular organelle for inflammatory phenotyping, establishing organelle level image analysis as a potential complement to molecular assays for immune cell classification.

The onset of mitosis in animal cells is marked by nuclear envelope breakdown (NEBD), which requires coordinated disassembly of nuclear pore complexes (NPCs) and the nuclear lamina. Polo-like kinase 1 (Plk1/PLK-1) initiates NEBD through phosphorylation of NPC components, but mechanisms that spatially regulate PLK-1 between NPCs and the nuclear interior are unclear. Here, we identify the Caenorhabditis elegans TMEM209 homolog NPP-28 as a transmembrane nucleoporin that concentrates PLK-1 at the nuclear envelope while limiting its nuclear accumulation to promote NEBD. We identify docking sites within NPP-28 that mediate PLK-1 nuclear envelope-association. NPP-28 is phosphorylated by PLK-1 in vitro, and mutational analysis suggests that NPP-28-mediated docking facilitates NEBD, whereas phosphorylation prevents premature NEBD. Simultaneous loss of npp-28 and the transmembrane nucleoporin npp-12/gp210 exacerbates mitotic NEBD defects in early embryos and causes severe defects in germline development. Together, our findings identify NPP-28 as a spatial regulator of PLK-1 during mitosis with functional relationships with other conserved transmembrane nucleoporins depending on context.

The sarcoplasmic reticulum (SR) is an elaborate membrane network and the primary intracellular Ca2+ store for muscle contraction. SR abundance, morphology, and spatial organization must be remodeled during muscle atrophy and hypertrophy, yet the mechanisms underlying this remodeling remain poorly understood. Autophagy has been implicated in SR homeostasis and may contribute to this process. We previously showed that a subset of Drosophila larval muscles remodels into adult dorsal internal oblique muscles through an autophagy-dependent process accompanied by extensive SR reorganization. Here, we show that autophagy is essential for maintaining SR abundance and architecture during muscle remodeling. Blocking autophagosome formation caused the accumulation of large aggregates of SR membranes, whereas blocking autophagosome-lysosome fusion led to the accumulation of autophagosomes with SR transmembrane proteins localized to their membranes. Moreover, disrupting LC3-interacting region (LIR)-dependent cargo recognition did not impair SR remodeling, arguing against a major contribution from selective autophagic degradation of the SR. Instead, our findings suggest that SR membrane components are incorporated into autophagosomal membranes during muscle remodeling. We propose that this membrane flow couples the removal of excess SR membrane to autophagosome biogenesis during atrophy, thereby facilitating large-scale SR reorganization during muscle remodeling.

Cell surface receptors are regulated via a variety of mechanisms including proteolytic inactivation by metalloproteases. Cells often co-express both the receptor and its cognate protease simultaneously on the cell surface, often with the proteases in an active state. Despite this, receptor cleavage is generally not constitutive, and rather must be induced. How a receptor and its inactivating protease can both be present in their active form on the cell surface with no cleavage of the target receptor is unclear. Using the efferocytic receptor MERTK and its inactivating protease ADAM17 as a model, along with super-resolution microscopy approaches, we demonstrate that MERTK and ADAM17 are localized to distinct sub-regions of the plasma membrane. These microdomains are distinct subcellular structures, with MERTK contained within actin-based corrals and ADAM17 contained within caveolae. Induction of MERTK cleavage by PMA results in the reorganization of the membrane-proximal actin cytoskeleton that enables MERTK to diffuse into ADAM17-containing microdomains. Only after localizing to ADAM17-containing microdomains is MERTK cleaved. These results demonstrate that the spatial organization of the plasma membrane is dynamically regulated, with reorganization of membrane microdomains playing a role in the regulation of ADAM17 activity.

WAVE and WASH polymerize Arp2/3-mediated branched actin at the leading edge of migrating cells and on the surface of endosomes, respectively. These two proteins are regulated within similar multiprotein complexes, the assembly mechanism of which remains poorly understood. Here we found using mass spectrometry that the two smallest subunits, BRK1 of the WAVE complex and CCDC53 of the WASH complex, interact with each other and with the assembly factor HSBP1. HSBP1 promotes WAVE and WASH assemblies and their respective activities, migration persistence and endosomal branched actin. Reduced levels of WAVE and WASH complexes caused by HSBP1 depletion can be corrected by providing cells with an excess of BRK1 or CCDC53 to assemble their specific complex. Unexpectedly, endogenous levels of BRK1 cross-regulate WASH assembly, whereas those of CCDC53 cross-regulate WAVE assembly. We found that various oligomers comprising BRK1, CCDC53 and HSBP1, including a heterotrimer containing one molecule of each, are formed through their promiscuous coiled coils. These oligomers play a coordinating role in the assembly of WAVE and WASH complexes.

Although whole-genome doubling (WGD) accompanies a drastic increase in chromosome number, post-WGD cells often continue to divide. How mitosis is sustained under increased chromosome number remains largely unknown. Here, we found that WGD cells require longer mitotic spindles than isogenic diploids to maintain spindle bipolarity. WGD increased mechanical stress on spindle poles during metaphase, associated with a higher risk of pole fragmentation. Disrupting kinetochore-microtubule attachment alleviated pole stress and fragmentation in WGD cells, suggesting that WGD-linked mechanical stress arises from spindle-chromosome interactions. Artificial spindle elongation reduced the pole stress and mitigated pole fragmentation defects in WGD cells. Our findings identify chromosome gain as a critical mechanical challenge to spindle organization and establish spindle size scaling as an adaptive mechanism that preserves mitotic fidelity after WGD.

WAVE is a major driver of lamellipodial protrusions. Previous studies suggested that Abelson (Abl)-dependent tyrosine phosphorylation of WAVE promotes actin polymerization and lamellipodia formation. Here, we provide evidence that Abl tyrosine kinase rather restricts lamellipodial localization of WAVE and inhibits lamellipodial cell spreading. Additionally, overexpression of a cytoplasmic PTP61F phosphatase increases WAVE localization at lamellipodia tips. PTP61F overexpression or pharmacological inhibition of Abl/Src kinases even protects cells from lamellipodia disruption induced by pervanadate, a potent protein tyrosine phosphatase inhibitor. By contrast, overexpression of a phosphatase-deficient PTP61F trapping mutant phenocopies loss of wave function. We identify the tyrosine Y234 as a prominent phosphorylation site in WAVE. Rescue experiments using CRISPR/Cas9-based KO wave mutant cells confirmed an important role of PTP61F-mediated WAVE dephosphorylation in lamellipodia formation. Our data suggest a model in which calpain-mediated proteolytic release of ER-bound PTP61F antagonizes Abl/Src-dependent WAVE phosphorylation and acts as a mechanism promoting re-localization of WAVE to lamellipodial tips.

Microtubules, composed of - and {beta}-tubulin polymers, are essential components of the eukaryotic cytoskeleton. They maintain cellular shape and structural integrity and play critical roles in cell division and in intracellular vesicular transport. In Plasmodium, the parasite that causes malaria, nuclear replication during the liver stage is among the fastest known in eukaryotic cells and relies heavily on microtubules for DNA segregation and cytoskeletal organization. Despite their importance, the role of microtubules in liver stage development remains largely unexplored. Here, we investigated microtubule dynamics during liver stage development using a combination of cell and molecular biology techniques, expansion microscopy, and live-cell imaging. By employing antibodies specific for {beta}-tubulin post-translational modifications (PTMs), we found that the Plasmodium sporozoites subpellicular microtubules (SSPM) persist during liver infection, giving rise to liver stage parasite microtubule bundles (LSPMB). These LSPMB form multimeric tubulin structures within hepatocytes and are redistributed to the hemi-spindle poles of parasite nuclei during schizogony. Deletion of the C-terminal region encompassing all known Plasmodium -tubulin PTM sites prevented sporozoite migration from the mosquito midgut to the salivary glands, effectively blocking parasite transmission. Using Plasmodium microtubule-specific depolymerisation drugs, we found that while LSPMB are stable in sporozoites, they exhibit dynamic behavior during hepatocyte infection. Given the regulatory role of PTMs in microtubule dynamics, we generated parasite mutants by substituting and deleting key {beta}-tubulin C-terminal residues involved in PTMs. Substitution of the polyglutamylation site with alanine and deletion of the C-terminal tyrosination/detyrosination motifs impaired parasite growth during liver infection. Together, our findings reveal extensive microtubule remodeling during liver stage development and establish -tubulin C-terminal modifications as critical regulators of both intracellular development and parasite transmission of Plasmodium parasites.

Anion exchanger 4 (Ae4), encoded by solute carrier family 4 member 9 (Slc4a9), is required for normal salivary secretion, but how it supports fluid secretion remains poorly understood. Nearly a decade ago, we developed a mathematical model that attributed reduced secretion following Ae4 knockout primarily to Ae4 mediated monovalent cation transport. Here, we reassess that interpretation by developing a dynamic model of salivary fluid secretion informed by subsequent experimental findings. The model incorporates {beta}-adrenergic regulation of Ae4 and an additional apical chloride conductance shared by the control and knockout. To isolate the contribution of Ae4, the NKCC1 and Ae2 cycle rates calculated from the evolving control state are also prescribed in the knockout. Under these conditions, the knockout model secretes 20% less fluid over 600 seconds despite identical apical chloride conductances. The control begins with a larger intracellular chloride store and continues to acquire chloride through Ae4 during stimulation. The intracellular chloride difference increases during stimulation, whilst the weaker apical electrochemical driving force in the knockout reduces chloride export and fluid secretion. The analysis also identifies the alkalinity requirement of sustained Ae4 transport and quantifies the additional NKCC1 and Ae2 transport needed to compensate for its loss. These results support intracellular chloride availability as a mechanism linking Ae4 loss to reduced salivary secretion.

Centriolar satellites are membrane-less organelles that store, modify and traffic proteins, in particular those of the centrosome and the primary cilium, and their deregulation is linked to ciliopathies, neurodevelopmental and neurodegenerative disorders. Satellite number, size, intensity and spatial distribution vary with cell type and cell state, yet satellites are usually quantified by a single readout, the pericentrosomal fluorescence intensity in maximum-projected images, which cannot distinguish changes in satellite number, size and position. Here we present SatelliteQ, an open-source Fiji and Python workflow for object-based quantification of satellites in 2D, 3D and time-lapse images. SatelliteQ segments satellites, detects centrosomes or primary cilia in the same cells, and reports satellite number, size, intensity, distance to the centrosome, shape and spatial distribution per satellite and per cell, with interactive graph and spatial viewers. Its measurements correlate closely with Imaris-based quantification; absolute values depend on segmentation settings and analysis mode, whereas the biological differences tested here were preserved across matched settings. Using SatelliteQ, we resolved distinct spatial signatures of satellite dispersal induced by microtubule depolymerization and by disruption of weak hydrophobic interactions, quantified differences between cell types and the scaling of satellite number with cell size, captured PCM1 granule dynamics in living cells, and compared satellite organization between ciliated and unciliated cells. Depletion of the ciliopathy-associated satellite protein CEP290 altered satellite abundance and pericentrosomal organization, while patient-derived cells carrying CEP290 mutations displayed divergent satellite phenotypes that were not explained by ciliation alone. SatelliteQ thus establishes satellite properties as a multidimensional cellular phenotype and provides an accessible framework for exploring satellites as an organelle-level readout in disease.

Cell-cell adherens junctions (AJs) and their dynamic cytoskeletal linkage power morphogenesis. AJs are enormous complexes with hundreds of proteins linked by multivalent interactions. Like other biomolecular condensates, intrinsically disordered regions (IDRs) in junctional proteins play important roles in AJ assembly and function, using spacer elements to span distances, and stickers to engage targets. To define molecular mechanisms, we need to define the functional units within IDRs. Drosophila Canoe, homolog of human Afadin, is our model. Canoe mediates morphogenesis and has an extensive IDR, with two conserved F-actin-binding stickers and two poorly conserved spacers. We combined biochemical, genetic and cell biological approaches to define the function of these IDR elements. While no single element is essential, deleting the full IDR essentially eliminates Canoe function. By scrambling the amino acid sequence of the spacers, we find that length and composition are more important than amino acid sequences, though sequences in the C-terminal spacer affect Canoe localization. Finally, we test redundancy of the F-actin-binding stickers. Deleting both reduces but does not eliminate viability, and sensitized assays reveal their redundant roles. These data reveal the robustness of IDRs.

Samp1 is an inner nuclear membrane protein required for myogenic differentiation and involved in chromatin organization at the nuclear periphery. Here, we investigated whether these functions are connected by studying the effects of Samp1 depletion during C2C12 myogenic differentiation using immunofluorescence microscopy, RNA sequencing, FRIC, and chromosome-positioning analysis. Samp1-depleted cells showed strongly reduced MyHC expression and virtually abrogated multinucleated fiber formation. Although cell-cycle withdrawal was not prevented, the transcriptional program driving differentiation was drastically perturbed, with reduced muscle-associated transcripts and incomplete repression of genes normally downregulated during myogenesis. Samp1 depletion also disrupted peripheral chromatin organization and prevented the accumulation of peripheral heterochromatin typically seen during differentiation. In addition, radial chromosome distribution was disrupted, evidenced by the failure of chromosome 8 to reposition to the nuclear periphery during differentiation. Together, these findings link the requirement for Samp1 in myogenic differentiation to its role in genome organization at the nuclear periphery.

Fluorescent proteins are widely used as quantitative reporters of protein abundance in living cells. Here we show that this relationship can break down under translation inhibition: the cellular ribosomal abundance can increase while fluorescence intensity remains unchanged or even decreases. Using fluorescent reporters of ribosome abundance in Escherichia coli and Bacillus subtilis, we find that cellular fluorescence intensity quantitatively tracks ribosome abundance, inferred by RNA-to-protein ratio, under nutrient-limited growth but that is no longer true during chloramphenicol treatment. In E. coli, the discrepancy occurs with transcriptional and translational reporters and with both ribosomal protein and ribosomal RNA promoters. To distinguish changes in fluorescence output per reporter from changes in reporter abundance, we constructed a fluorescent protein-LacZ dual reporter in which fluorescence and an independent enzymatic estimate of reporter abundance are obtained from the same protein. Under translation inhibition, LacZ-derived reporter abundance increases whereas fluorescence intensity remains approximately constant, showing that fluorescence output per unit reporter abundance decreases. In a companion study, Bakshi and colleagues demonstrate that the effect extends to other fluorescent proteins and other translation inhibitors, indicating that it is not specific to a particular fluorescent protein or translation inhibitor. Our results show that fluorescent-protein calibration can be condition-dependent and cannot be assumed to transfer across physiological perturbations.

Meiotic chromosome segregation requires the earlier coordination of chromosome morphogenesis with cell-cycle progression. In C. elegans, the remodeling of chromosomes in preparation for the meiotic divisions and oocyte maturation is regulated by the master cell-cycle regulator cyclin-dependent kinase 1 (CDK-1). Here, we identify CYB-4 as a meiosis-specific B-type cyclin that is required for CDK-1-dependent functions in meiotic prophase. In a genetic suppressor analysis of a mutant defective in chromosome axis assembly, we isolated a null allele that revealed a function for CYB-4 in regulating chromosome structure at two key prophase transitions. Loss of CYB-4 first delays meiotic entry and the onset of chromosome axis formation, and at pachytene exit delays chromatin condensation, bivalent remodeling, and oocyte maturation. The defects in timely CO formation and bivalent packaging that accompany these delays are ultimately resolved before prophase exit, indicating that CYB-4 is required for the scheduling of events rather than if they occur. Together, our findings provide evidence that CDK-1 activity during meiotic prophase is defined by CYB-4 and support a model in which CYB-4/CDK-1 are essential for the sharp transitions at prophase entry and pachytene exit that coordinate chromosome morphogenesis with meiotic progression.

Tropomyosins play a central role in the regulation of the actin cytoskeleton by assembling along filaments into rope-like, head-to-tail polymers ("clusters") along filaments that regulate the binding and activity of numerous actin-binding proteins. Importantly, in cells, tropomyosins do not decorate actin filaments uniformly; some filaments are devoid of tropomyosins, whereas others recruit specific isoforms. To explore the diversity of tropomyosin-isoform binding kinetics and identify factors that control their assembly, we designed tropomyosin constructs, bearing a natural N-terminal acetylation and targeted cysteine mutations for fluorescence labelling. We analysed the binding of different tropomyosin isoforms to individual actin filaments reconstituted inside a microfluidic chamber. We show that, despite significant differences in spontaneous nucleation and elongation rates, all tested tropomyosin isoforms can coassemble within the same clusters. In addition, we identified tropomodulin and leiomodin, two pointed-end-binding proteins, to efficiently nucleate the formation of tropomyosin clusters on actin filaments, for all tested tropomyosin isoforms. Notably, while tropomodulin nucleates tropomyosin clusters from the pointed end, leiomodin does so along the entire filament.