Microtubules are core cytoskeletal polymers that support epithelial organization, polarity, and tissue morphogenesis. They are composed of α- and β-tubulin heterodimers encoded by multigene families that generate closely related but distinct isotypes. Although several tubulin isotypes have been implicated in ocular development and disease, their spatial organization during corneal morphogenesis remains poorly defined. Here we establish a resource visualizing and quantifying tubulin isotype localization during corneal development using the chick embryo. Comparative amino acid sequence analysis demonstrates that chick and human α- and β-tubulin isotypes are highly conserved within structural and catalytic domains, with divergence concentrated in C-terminal regions normally associated with post-translational modification sites. Relating sequence features to tissue organization, we performed longitudinal immunohistochemical analysis of five tubulin isotypes across corneal development. These analyses reveal distinct and dynamic patterns of isotype enrichment along apicobasal axes, as well as stage-dependent redistribution during epithelial maturation and endothelial differentiation. TUBA5/TUBA4A displays particularly restricted localization, including enrichment in migratory corneal stromal progenitors and the developing corneal epithelium and endothelium. Together, these data provide a conserved spatial framework for tubulin isotype organization during corneal morphogenesis and establish the chick embryo as a tractable model for investigating isotype-specific microtubule organization during epithelial development.

Cilia are highly conserved organelles that use hundreds of unique proteins to drive extracellular motility and serve as signaling hubs for eukaryotic cells. The Ciliogenesis and Planar Polarity Effector (CPLANE) protein complex controls basal body docking during early ciliogenesis and recruits the intraflagellar transport (IFT) proteins to the base of growing cilia. Leveraging recent advantages in deep learning neural networks and high-performance computing, we predict the structure of JBTS17, the largest CPLANE subunit, and the tertiary structure of the complete CPLANE complex, revealing direct contacts between JBTS17 and three other CPLANE members. We then used this model for an in silico protein-protein interaction screen that identified several novel JBTS17 interactors. Experiments in Xenopus confirmed a functional link in vivo between Jbts17 and the Spinocerebellar ataxia-associated protein TTBK2. The data provide new insights into mechanisms of CPLANE function during ciliogenesis and demonstrate the power of modeling protein complexes for the discovery of physical and functional protein interactions in cell homeostasis and disease.

Bin/Amphiphysin/Rvs (BAR) domain-containing proteins participate in membrane-remodeling processes, including vesicle trafficking, cell division, and cell migration. We previously demonstrated that ciBAR1 interacts with Cby1 and colocalizes at the ciliary base to promote ciliogenesis. However, the function of the Cby1/ciBAR1 complex remains poorly understood. Here, using recombinant proteins and reconstituted liposomes, we show that ciBAR1 alone exhibits little or no lipid-binding activity, whereas Cby1 promotes ciBAR1 binding to liposomes. In nanobar assays, Cby1 also promotes ciBAR1 membrane association and enrichment at regions of high membrane curvature. The Cby1/ciBAR1 complex preferentially associates with liposomes containing PI(4,5)P2 and PI(3,4,5)P3, two phosphoinositides (PIs) enriched near the ciliary base. Consistent with these findings, ciBAR1 mutants defective in either membrane or Cby1 binding fail to localize to the ciliary base and are unable to rescue ciliogenesis defects in ciBAR1-KO NIH3T3 cells. Furthermore, ultrastructural analysis reveals that ciBAR1-KO cells exhibit defects in preciliary vesicle recruitment and an accumulation at the ciliary vesicle stage, suggesting impaired progression to ciliary membrane extension and ciliary bud formation. Collectively, our findings support a model in which Cby1 potentiates ciBAR1 binding to PI-enriched membranes at the basal body, where the Cby1/ciBAR1 complex mediates membrane-associated processes during early ciliogenesis.

The deregulation of neural stem cell (NSC) homeostasis underlies neurodevelopmental disorders and tumorigenesis. In the Drosophila larval brain, NSCs divide asymmetrically along an invariant apical-basal axis to yield a larger self-renewing stem cell and a smaller, differentiating sibling. NSC divisions are instructed by centrosomes, which recruit pericentriolar material (PCM) important for microtubule-organizing activity. During interphase, NSC centrosomes are defined by asymmetries in PCM composition and functional activity. These asymmetries help align the mitotic spindle and position the cleavage furrow. Here, we show that the conserved RNA-binding protein Orb2 is required to enrich Polo kinase, the master regulator of centrosome maturation, at centrosomes throughout the cell cycle. Loss of Polo correlates with compromised localization of downstream PCM effectors in orb2 NSCs. Despite these impairments, orb2 NSCs build and orient their spindles normally to complete asymmetric cell division. However, through live imaging and clonal analysis, we demonstrate that Orb2 is required to maintain proper cell size, as loss of orb2 yields significantly smaller NSCs and sibling cells. We further show that Orb2 supports the timely clearance of cortical myosin important for cleavage plane positioning. These data indicate that Orb2 promotes NSC centrosome maturation and size of the neural progenitors. [Media: see text] [Media: see text] [Media: see text] [Media: see text] [Media: see text] [Media: see text] [Media: see text] [Media: see text].

The nuclear pore complex (NPC) mediates nucleocytoplasmic transport while contributing to nuclear organization and genome regulation. In Saccharomyces cerevisiae, the nucleoporin Nup170, positioned in the NPC's inner ring, plays a critical role in subtelomeric gene silencing, telomere tethering to the nuclear envelope, and association of silencing factors with subtelomeric chromatin. However, the molecular basis of these functions and their relationship to NPC architecture remain unclear. Here, we define a nucleoplasm-exposed region of Nup170 required for subtelomeric gene silencing. Systematic truncation analysis reveals that Nup170 residues between ∼990 and 1200 are necessary to restore silencing, telomere positioning, and subtelomeric gene expression in nup170Δ cells. Mapping these truncations onto high-resolution structural models of the NPC places this region on a nucleoplasmic-facing surface of the inner ring, accessible through openings in the outer ring scaffold. Targeted single amino acid substitutions within this region, including nup170[D1099G], phenocopy the subtelomeric silencing defect, telomere tethering defect, and reduced SIR complex binding of nup170Δ. These results demonstrate that a short, structurally defined surface of Nup170 within the NPC scaffold directly supports epigenetic regulation at the nuclear periphery. Our findings reveal how architectural nucleoporins perform specific genome regulatory functions through discrete interfaces, highlighting functional specialization among individual NPCs.

While lysosomes are typically globular in morphology, lipopolysaccharide-activated macrophages reorganize lysosomes into an expanded tubular network. Here, we sought to determine if the V-ATPase and the lysosomal pH contribute to LPS-mediated lysosome tubulation in macrophages. We found that inhibition of the V-ATPase prevented lysosome tubulation and collapsed preformed tubules. However, the V-ATPase also controls mTORC1 activity, which itself is needed for tubulation. To distinguish between lysosomal pH and mTORC1, we turned to NH4Cl, which alkalinized lysosomal pH but did not interfere with mTORC1; yet NH4Cl blocked tubulation showing that an acidic lysosomal pH is needed for lysosome remodelling. Moreover, clamping the pH to either acidic or alkaline pH caused tubules to collapse, indicating that the pH gradient promotes tubulation, rather than a specific pH. These effects were not due to altered microtubule organization or impaired lysosome motility, suggesting that motors remained associated with lysosomes. On the other hand, while LPS did not alter the average pH of spherical or tubular lysosomes, growing tubules displayed a more acidic peripheral end relative to the pericentral end. Based on this observation, we propose that a localized pH gradient along the tubule may enable tubulation by modulating factors that catalyse tubule growth. [Media: see text] [Media: see text] [Media: see text] [Media: see text] [Media: see text] [Media: see text].

Quantitative interpretation of single-molecule localization microscopy (SMLM) data remains limited by fluorophore blinking, incomplete or variable labeling, and the lack of calibration standards that are tunable, synthetic, and compatible with cellular imaging. Here, we evaluate self-assembling protein nanocages as stoichiometric benchmarks for copy-number calibration in STORM imaging. We generated mammalian expression constructs encoding nanocages containing 60- or 120-copies of different GFP variants and repositioned the fluorescent protein to the N-terminus to improve accessibility for anti-GFP nanobody labeling. Among the variants tested, mCitrine-tagged nanocages showed the most consistent expression and labeling with commercially available AF647-conjugated anti-GFP nanobodies. Using an analysis pipeline that matched widefield-detected nanocages to STORM-localized clusters, we found that mCitrine 120-mer nanocages produced localization distribution in which the mean of the distribution was shifted by approximately two-fold compared to the 60-mer. These results suggest that optimized protein nanocages can serve as useful calibration standards for STORM in the moderate to high copy-number range. More broadly, this approach provides a flexible framework that could be extended to additional nanocage stoichiometries, labeling strategies, and SMLM modalities.

The B cell receptor (BCR) drives the differentiation of naive B cells into activated plasma cells that produce antibodies. The BCR consists of a plasma membrane-bound surface immunoglobulin (sIg) that binds antigens, and two coreceptor subunits CD79A and CD79B. The intracellular domains of CD79A and CD79B initiate immune signaling and endocytosis of the receptor in healthy B cells. In diffuse large B cell lymphoma (DLBCL), an aggressive form of human blood cancer, the activated B cell like (ABC) subtype exhibits constitutive signaling that drives survival and proliferation. Here, in a model of human ABC DLBCL, we determine the localization of BCR components relative to plasma membrane structures using correlative super-resolution light and platinum replica transmission electron microscopy. We find that spontaneous clusters of the surface Immunoglobulin common to ABC lymphoma localize to smooth raised membrane domains. These structures are involved in the endocytosis of large receptor clusters. Surprisingly, activated phosphorylated CD79A (pCD79A) shows little colocalization with clathrin on or off smooth raised membranes. Furthermore, pCD79A spatially segregates away from both the slg and CD79B subunits of the BCR complex. A similar distribution was found for downstream phosphorylated Src family kinases. We propose that a disengagement of pCD79A away from sIg and CD79B allows the receptor to evade down-regulation through endocytosis and maintains signaling at the plasma membrane. This mechanism could drive aberrant signaling and the proliferation of lymphomas leading to disease.

The Cancer Dependency map project (DepMap) has created an unprecedented resource of siRNA and CRISPR genetic screens across more than 1000 cell line models. Numerous computational tools have been developed to analyze DepMap outputs and have revealed new genetic dependencies relevant to cancer. Here we extracted correlation information from the DepMap using curated gene sets of biological pathways to determine if this approach could reveal new high confidence candidates. Beginning with a set of curated DNA repair proteins we determined correlations of query gene knockout fitness across all DepMap cell lines. As expected, this approach extracted many known genome stability genes and also suggested several additional candidates not previously linked to genome maintenance. To validate our analysis we used siRNA depletion for candidate genes and identified several factors whose depletion increases DNA damage or delay repair. Among these was WDR89. WDR89 nucleolar intensity increases in an ATM dependent manner upon DNA damage and WDR89 physically interacts with a number of nucleolar proteins. Depletion of WDR89 leads to accumulation of p53 in the nucleus, p21 accumulation and G2/M cell cycle arrest 24 hours post-irradiation. These data establish WDR89 as a new genome stability regulator and highlight the value of DepMap for predicting previously unknown biology.

Septins are cytoskeletal proteins that regulate cytokinesis in fungi and animals, yet their functions in choanoflagellates-the closest living relatives of animals-have remained unknown. Salpingoeca rosetta, a choanoflagellate that switches between unicellular and multicellular forms, encodes four septins that are closely related to animal and fungal septins. Disruption of three of these septins altered cell size and impaired multicellular rosette development, with two of these three also required for normal rosette integrity. Characterization of Sros_septA, which showed the strongest phenotype, revealed a role in cytokinesis: mutant cells underwent furrow ingression but frequently failed to complete cell division, resulting in enlarged, multinucleated cells. Cell size and rates of cytokinesis failure and multinucleation were elevated in uninduced Sros_septA mutant cells and increased further upon rosette induction, revealing a heightened requirement for Sros_septA in cytokinesis during multicellular development in S. rosetta. Consistent with these cytokinesis defects, endogenously tagged Sros_SeptA dynamically redistributed from the basal pole in interphase cells to the cleavage furrow and nascent intercellular bridge during cell division. These findings identify roles for septins in cytokinesis and multicellular development in S. rosetta and offer a framework for exploring the evolution of cytokinetic regulation alongside the emergence of multicellularity in the animal stem lineage. [Media: see text] [Media: see text] [Media: see text] [Media: see text] [Media: see text].

Endosomal Sorting Complexes Required for Transport (ESCRT) machinery drives membrane remodeling and scission events essential for multivesicular body (MVB) biogenesis. The accessory ESCRT-III subunit Vps60 (mammalian CHMP5) shares structural features with the major polymerizing subunit Snf7, including the capacity to form homopolymers in vitro. Here, we demonstrate in Saccharomyces cerevisiae that Vps60 recruitment and polymerization require conditions that permit Snf7 polymer turnover: Vps60 is largely excluded when Snf7 assembly is absent and when Snf7 disassembly is blocked, consistent with recruitment of Vps60 late in the ESCRT-III cycle to Snf7 assemblies that undergo normal turnover. Structure-function analysis reveals that Vps60 regulation diverges fundamentally from core ESCRT-III subunits: while occlusion of the Vps60 C terminus by a GFP tag drives constitutive membrane association and polymer assembly, C-terminal truncation impairs endosomal recruitment, indicating that the Vps60 C terminus is required for membrane recruitment, in contrast to the autoinhibitory C-terminal domains of core ESCRT-III subunits. A C-terminally tagged Vps60 that constitutively associates with membranes phenocopies loss of Vps60 function in both intralumenal vesicle (ILV) formation and cargo sorting. These findings indicate that despite structural similarity to Snf7, Vps60 functions as a late-arriving accessory factor whose recruitment and assembly are tightly coupled to Snf7 dynamics. [Media: see text] [Media: see text] [Media: see text] [Media: see text].

The TORC1 (Target of Rapamycin Complex 1) kinase complex coordinates eukaryotic cell growth and metabolism in response to nutrient availability. In yeast, one of the proteins under TORC1 control is the Npr1 kinase mediating nitrogen control of several plasma membrane transporters, yet the metabolic signals governing this pathway remain poorly understood. Here, we first investigated the previously described and unexpected activation of TORC1-Npr1 signaling upon acute glucose starvation. We show that this response occurs only under specific nitrogen conditions, notably in cells grown on proline or glutamine, and depends on increased ATP production driven by stimulated respiration. In proline-grown cells, this respiration is fueled by catabolism of α-ketoglutarate, while high α-ketoglutarate levels negatively affect TORC1-Npr1 activation. We next analyzed TORC1-Npr1 activation following NH4[+] addition to proline-grown cells. Our results confirm that glutamine accumulation is not required for this activation and instead show that NH4[+] assimilation is associated with a decrease in α-ketoglutarate levels and a sustained increase in ATP production, concomitant with TORC1-Npr1 activation. In cells lacking the NH4[+]-assimilating glutamate dehydrogenases, both increased ATP production and TORC1-Npr1 activation are impaired. Together, these findings identify ATP and α-ketoglutarate as important metabolic determinants of TORC1-Npr1 signaling in response to nitrogen availability.

Z-discs define sarcomere boundaries and anchor actin filaments in muscle, yet many structural components remain uncharacterized. In this paper, we identify three new members of the Alp/Enigma family in Drosophila, including Uchmaz (CG42319), a PDZ domain-containing protein, that we characterize in detail. We used CRISPR/Cas9 to generate multiple uchmaz knockout alleles, which are viable but result in a flightless phenotype. Mutants exhibit disrupted sarcomere organization in the indirect flight muscles (IFMs), including fragmented and misaligned Z-discs and reduced accumulation of α-Actinin and other Z-disc markers. We find that Uchmaz localizes to the Z-disc, associates with α-Actinin, and is dependent upon α-Actinin for its localization to the Z-disc. These results identify Uchmaz as a component of IFM myofibril architecture required for muscle function. Since a human ortholog of uchmaz, PDLIM2, is associated with severe muscle dysfunction in muscular dystrophies and myofibrillar myopathy, our studies provide insight into conserved mechanisms of Z-disc assembly and its relevance to human muscle disease.

Abnormal α-synuclein (α-syn) accumulation and mitochondrial dysfunction are central features of Parkinson's disease (PD), and increasing evidence suggests that these processes are closely connected. Using yeast models to manipulate mitochondrial metabolic states, we investigated how mitochondrial activity may affect α-syn accumulation and toxicity. Under the fermentative growth condition, we found that α-syn localized predominantly to the plasma membrane and was non-toxic. Respiratory growth induced cytoplasmic α-syn accumulation, mitochondrial abnormalities, and marked toxicity dependent on α-syn expression and membrane binding. Surprisingly, genetic disruption of mitochondrial respiration also enhanced α-syn toxicity despite causing little α-syn accumulation, indicating that both increased demand of mitochondrial respiratory function and compromised respiratory capacity can sensitize cells to α-syn toxicity. Overexpression of HAP4, a master regulator of mitochondrial biogenesis and respiratory gene expression, suppressed α-syn toxicity, but this protection required functional mitochondria. Together, these findings demonstrate that distinct mitochondrial functional states exert markedly different effects on α-syn toxicity and reveal a complex relationship between mitochondrial function and cellular response to α-syn expression. These models provide a new system for investigating how changes in mitochondrial function contribute to α-syn-induced cellular stress.

Dynactin, a complex essential for cytoplasmic dynein function, contains an Arp1 mini-filament with a pointed-end sub-complex including Arp11 and a shoulder sub-complex including p50 and p150. We recently identified in Aspergillus nidulans a vezatin homologue, VezA, that enhances dynactin assembly, but VezA does not co-localize with dynactin, suggesting a transient interaction. It was found that overexpression of p50 separates the shoulder from the mini-filament and causes late-onset neurodegeneration in mice, but its cellular effects in the context of VezA need to be studied. Here, we surprisingly found that p50 overexpression in A. nidulans causes Arp11, p50 (but not p150), and VezA to form co-localized clusters whose presence depends on Arp1. Formation of the VezA cluster also depends on Arp11, and VezA significantly enhances the intensity of Arp11-GFP clusters but not that of p50-GFP clusters. Moreover, an evolutionarily conserved region of p50 (aa17-25) forming a beta-sheet structure with Arp1, as revealed in previous cryo-EM studies, is critical for the formation of Arp11, p50, and VezA clusters. These results suggest that excess p50 induces cluster formation driven by p50-Arp1 interactions, and that this assembly intermediate lacking the intact shoulder traps VezA, which participates in the Arp11-Arp1-p50 interactions.