Journal of Cell Biology

The Golgi-localized, {gamma}-ear containing, ADP-ribosylation factor binding proteins (GGAs) are a family of adaptor proteins that regulate transport of specific cargo receptors from the Golgi to endosomes. For many years it was assumed that GGAs transport cargo via interaction with the adaptor complex AP-1. However, recent findings suggest that GGA and AP-1 may have opposing roles, with GGAs facilitating forward transport between Golgi and endosomes, and AP-1 mediating the opposite trafficking step. To shed light on the functional connection of GGAs with AP-1, we combined CRISPR-Cas9 gene editing with live-cell imaging and TurboID-based proximity labelling. We find that GGAs localize not only to the Golgi apparatus but also, to a greater extent, to peripheral ARF1-positive compartments responsible for secretory trafficking and endocytic recycling. At both, the Golgi and peripheral sites, we observe distinct sorting domains containing either AP-1 or GGAs alone, as well as domains in which both adaptors are present. Interestingly, GGAs can recruit clathrin lattices independently of AP-1. Proximome mapping shows that AP-1 specific cargoes only localize to AP-1 domains in the absence of GGAs. These findings point to a regulatory role of GGAs in AP-1 transport. We speculate that GGAs prevent binding of AP-1 to its cargo clients to avoid premature retrieval and to modulate bi-directional trafficking between the Golgi and endosomes.

The autophagy core machinery mediates the enclosure of cytosolic cargo destined for degradation in the lysosome. The Atg9-Atg2-Atg18 complex coordinates phagophore expansion via directed lipid transfer until closure of the phagophore rim. Using an Atg2 variant (Atg2-PM4) as a model of decelerated autophagosome biogenesis, we visualized the morphological states prior to autophagosome closure by cryogenic correlative light and electron microscopy in S. cerevisiae. Using in situ cryo-electron tomography, we find an enlarged rim morphology of an expanding phagophore in Atg2-PM4 cells in comparison with Atg2 wildtype condition. Analysis of segmented rim membrane features as well as surrounding and attached vesicles suggest that the enlarged rims are a result of cytosolic vesicles fusing with the growing phagophore. High-resolution imaging in this study shows that, apart from the initial nucleation phase, vesicle fusion can also contribute to phagophore expansion during later stages of autophagosome biogenesis.

The tight junction (TJ) protein cingulin binds directly to nonmuscle myosin 2B (NM2B) through sequences in its C-terminal rod-tail region and recruits it to tight junctions (TJ) to control membrane cortex mechanics, epithelial morphogenesis and cingulin conformation. However, the minimal sequence required for cingulin-NM2B interaction and how this interaction is regulated is not known. Here we identify a 19-aminoacid sequence at the hinge between the cingulin rod and tail that is required for cingulin-NM2B interaction, and we investigate the role of phosphorylation of Ser residues within this region in regulating this interaction. Immunofluorescence microscopy localization of NM2B in cingulin-KO cells rescued with mutant cingulin constructs shows that phospho-mimetic but not dephospho-mimetic cingulin mutants inhibit NM2B recruitment to junctions and downstream regulation of cingulin conformation and TJ tortuosity, correlating with cingulin-NM2B interaction, as determined by GST pulldown analysis. In contrast, either phospo-or dephospho-mimetic mutants of Ser residues within the cingulin head domain do not affect either NM2B recruitment to TJ, or cingulin conformation and localization in cells, or TJ membrane tortuosity. Finally, Ser residues within the hinge display the consensus sequence for protein kinases CK1 and CK2, and, through in vitro phosphorylation, site mutation analysis and use of inhibitors, we identify a complex interplay between CGN phospho-sites, with a prominent negative role of Ser1162 phosphorylation in the regulation of cingulin-NM2B interaction. In summary, we show that cingulin-NM2B interaction is regulated by cingulin phosphorylation within the hinge and identify a potential role for CK1 and CK2 kinases in cingulin phosphorylation.

Microtubule networks are major determinants of cell architecture and logistics. Microtubule organization and density are regulated by severing enzymes, which cut microtubule lattices or affect their growth and shortening. These activities can lead to microtubule amplification or disassembly, depending on the presence of microtubule stabilizers or destabilizers, but the interplay between these factors is poorly understood. Here, we reconstituted in vitro the activity of microtubule severase katanin together with microtubule minus-end stabilizers CAMSAPs, their binding partner WDR47 and microtubule depolymerase kinesin-13/MCAK. We confirmed that katanin can amplify or destroy microtubules in a concentration-dependent manner. CAMSAPs recruit katanin to microtubules and reduce katanin concentration needed for both amplification and destruction, whereas kinesin-13 completely abolishes microtubule amplification. WDR47 binds to microtubules decorated by CAMSAPs and suppresses katanin binding and severing. In addition, both katanin and WDR47 inhibit polymerization of CAMSAP-decorated microtubule minus ends. These data explain how these proteins act together to fine-tune microtubule minus-end stability without strongly increasing microtubule abundance. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=169 SRC="FIGDIR/small/714132v1_ufig1.gif" ALT="Figure 1"> View larger version (20K): org.highwire.dtl.DTLVardef@746fe3org.highwire.dtl.DTLVardef@5dd5a8org.highwire.dtl.DTLVardef@762373org.highwire.dtl.DTLVardef@1192db_HPS_FORMAT_FIGEXP M_FIG Graphical abstract C_FIG

Lipid droplets (LDs) are dynamic organelles that store neutral lipids and form in the endoplasmic reticulum (ER) membrane. Formation of new LDs is a controlled process and requires proteins with specific functions to form and grow from the ER membrane without any defect. In vitro studies have suggested a role for membrane curvature in LD emergence from the ER. Here, we use the membrane-shaping protein Pex30 to investigate the impact of ER membrane curvature on LD biogenesis and morphology. We modified the reticulon homology domain (RHD) of Pex30, which is responsible for tubulating the ER membrane, by extending the short hairpin transmembrane domains (TMD). The Pex30 (TMD) mutants cannot tubulate the ER membrane and generate less local membrane curvature that WT Pex30. Additionally, these mutants are unable to restore delayed LD biogenesis observed in cells devoid of Pex30. Our results indicate that Pex30 RHD generates local membrane curvature at ER subdomains that drives formation of new LDs.

Microtubules, stiff rods built up from tubulin dimers, form a cytoskeletal network whose structure, behaviour, and function have been extensively investigated, mainly from a mechanical perspective. Here, we describe a role for tubulin in the cellular stress response. We overexpressed tubulin dimers in a controlled fashion in 293F cells. Despite the engagement of autoregulation, a mechanism that degrades tubulin-encoding mRNAs when tubulin levels are high, a surplus of tubulin and microtubules is detected in overexpressing cells. This leads to altered microtubule behaviour, mitotic problems, deregulation of the cell cycle, and replication stress. Surprisingly, we also observe proteostasis defects in tubulin overexpressing cells, which we attribute to mitochondrial stress-related translation attenuation. Conversely, tubulin and microtubules are downregulated as part of the response to oxygen or glutamine deprivation. Together, our data link tubulin levels, and hence autoregulation, to cellular quality control and proteostasis. We propose that competitive interactions with key partners, including the mitochondrial protein import and general translation machinery, underlie the tubulin-mediated control of cellular homeostasis.

Rho family GTPases are central hubs in the signaling and cytoskeletal networks that govern cell morphology and behavior. GTPase-activating proteins (GAPs) inactivate them by accelerating GTP hydrolysis. However, a systematic analysis of GAPs in cell migration is lacking. Here, we report screens for RhoGAP expression and function in migratory Drosophila border cells. Constitutively active Cdc42, Rac, or Rho causes defects, demonstrating that negative regulation is critical. Integrating single-cell RNAseq with published datasets reveals that most of the 22 RhoGAPs are expressed in border cells. RNAi knockdown shows most RhoGAPs are functionally required. We developed automated image analysis tools to sensitively and objectively classify border cell morphologies, defining a normal morphological phase space. RhoGAP perturbations push clusters outside this range. In-depth analysis of RhoGAPp190 reveals that loss-of-function resembles Rho hyperactivation and gain-of-function resembles myosin II inhibition. Thus, complex spatiotemporal sculpting of RhoGTPase activities requires diverse RhoGAPs within a single cell type to control morphology and motility in vivo.

The intracellular cilia assembly pathway is a complex, multistep process that requires the continuous and coordinated incorporation of membrane material. However, how membrane remodeling occurs during early ciliogenesis is not yet understood. Moreover, the identity of the organelle(s) that supply membrane material for the nascent cilium has yet to be determined. Here, we extend the current model of primary cilia formation by showing that randomly attached distal appendage vesicles and tubules fuse laterally to generate a doughnut-shaped membrane structure. Centripetal fusion events follow to close the central hole. Our data demonstrate that both the Golgi apparatus and endocytotic pathways independently contribute to ciliogenesis. We identify the endocytotic protein GRAF1 as being essential during the early stages of ciliogenesis and for the delivery of plasma membrane-derived material to the developing ciliary membrane. Our three-dimensional ultrastructural analysis uncovers previously unrecognized intermediate stages in the intracellular cilia assembly pathway with GRAF1 as a novel regulator of ciliogenesis.

Motile cilia on eukaryotic cell surfaces are exposed to a wide array of external stresses. Ciliary remodeling in response to stress remains understudied despite being pivotal to cell homeostasis. Here, we apply a commonly used calcium perturbation to mimic hyper-osmotic cellular stress in the model ciliate Tetrahymena thermophila. We find that partially deciliated Tetrahymena, upon calcium shock, internalise entire ciliary axonemes into ring-like configurations we refer to as c-rings. Partially deciliated cells recover and regenerate new cilia over time. We propose that the recycling of c-rings releases axonemal building blocks to allow ciliary regeneration. We use time-course ultrastructure expansion microscopy (U-ExM) to show that the ciliary tubulin code undergoes a dynamic pattern of erasure during the bulk disassembly of doublet microtubules within the c-rings. Using whole cell quantitative proteomics, we find that partial deciliation induces the autophagy of c-rings and activates de novo dynein synthesis by upregulating several axonemal dynein assembly factors (DNAAFs). Transmission electron microscopy (TEM) and confocal imaging confirms that intact ciliary axonemes are encapsulated within VPS13A +ve autophagic vacuoles. We propose that internalised motile cilia undergo a regulated process of bulk degradation we term "motile ciliophagy" for the recycling of axonemal components for cilia regeneration. Our work establishes a link between ciliary turnover via macroautophagy and cellular homeostasis which may be a conserved response to stress.

Synaptic neurotransmission imposes high demands on membrane turnover, metabolism, and the remodeling of presynaptic molecular composition. While the impact of autophagy on neurotransmission has been firmly established, evidence for activity-dependent synaptic induction of autophagy remains surprisingly limited. Here, we demonstrate that amphisomes containing BDNF/TrkB are formed at presynaptic boutons following sustained synaptic activation. Activity-dependent bulk endocytosis serves as a membrane source for amphisome biogenesis, while key autophagy proteins are recruited to the active zone, and autophagy initiation is triggered locally by the energy-sensing kinase AMPK. BDNF/TrkB-containing amphisomes contribute to the turnover of key presynaptic cytoskeletal proteins involved in synaptic vesicle clustering. The formation of amphisomes following sustained synaptic activity facilitates both the degradation of these proteins and their replenishment through local translation of their mRNAs at presynaptic boutons. We propose that activity-induced synaptic autophagy largely reflects amphisome formation, which in turn is required for the replacement of proteins within the local presynaptic cytomatrix.

The neuronal endoplasmic reticulum (ER) extends from the soma into axons and dendrites to coordinate protein trafficking, lipid metabolism, inter-organelle organization, and calcium homeostasis. Conserved genes involved in shaping the tubular ER are implicated in neurodevelopment and neurodegeneration, suggesting that ER structure and dynamics influence neuronal health and drive pathogenesis. However, the links between ER morphology and neuronal function and resilience remain incompletely understood. While models typically depict the neuronal ER as a fully continuous network, here we show that micron-scale ER discontinuities in neurites are unexpectedly common in young, unstressed C. elegans. These discontinuities occur in both axonal and dendritic compartments with a consistent frequency that varies between motor and mechanosensory neuron types. Using live imaging and photokinetic assays of endogenously tagged markers of the ER, we confirm that these gaps reflect true loss of ultrastructural continuity. Subpopulations of ER tubule tips are highly motile, and the majority of ER discontinuities are resolved in less than an hour. Suggesting the formation of discontinuities is linked to cellular damage, their frequency increases with both age and environmental stress. Finally, in agreement with prior observations across models, discontinuities are exacerbated by impairment of certain ER shaping factors involved in hereditary spastic paraplegia, such as reticulon. These findings reveal a model where ER discontinuities are not uncommon in healthy animals, and provide a tractable system in C. elegans to dissect the molecular mechanisms maintaining ER structural homeostasis in vivo.

Actin cytoskeleton networks exhibit specialized architectural properties for specific cellular tasks, as determined by the actin-binding proteins (ABPs) associated with each network. Proper allocation of a limiting pool of actin monomers also helps shape the assembly of different F-actin networks. The ABP capping protein (CP) modulates F-actin network architecture through regulation of actin filament length by capping filament barbed ends. Using a combination of in vitro biochemistry and quantitative live-cell imaging, we characterize CP as a major regulator of inter-network competition between filopodia and mini-comets, two F-actin networks in the one-cell C. elegans embryo (zygote). We establish that this regulation is facilitated in part by competition for binding barbed ends between CP and the F-actin elongator formin CYK-1. Together, these results reveal a role for CP in determining F-actin network architecture and dynamics, regulating the coordination between actin assembly factors to assemble and maintain different dynamic F-actin networks, and allocation of G-actin between competing cortical F-actin networks. Summary for table of contentsCells assemble diverse actin cytoskeleton networks within a common cytoplasm for essential cellular processes. Yde et al. establish a role for Capping Protein, a regulator of actin filament length, in coordinating the balanced assembly of distinct actin networks in the C. elegans zygote.

Lipid droplets (LDs) accumulate in response to diverse cellular stresses. However, their regulation and physiological roles remain poorly understood in most contexts. Here, we show that, in budding yeast, chronic hyperosmotic stress induces sustained LD accumulation. Unlike the transient LD response observed during acute osmotic shock, chronic stress triggers prolonged, Dga1-dependent triacylglycerol synthesis. In the absence of triacylglycerol synthesis cellular fitness is severely affected. Lipidomic profiling reveals extensive membrane remodelling during chronic hyperosmotic stress, most notably a shift from phosphatidylethanolamine to phosphatidylcholine. In LD-deficient cells, the stress-induced PC increase is blunted and manipulation of PC synthesis modulates the fitness of triacylglycerol-deficient cells under hyperosmotic stress. Thus, LD accumulation and phospholipid remodelling underlie an adaptive response to chronic hyperosmotic stress. SummaryThis work demonstrates that membrane remodelling occurs in cells experiencing chronic hyperosmotic stress. Both triacylglycerol and phosphatidylcholine levels are increased. Cell fitness depends upon increased triacylglycerol synthesis and is further modulated by manipulating phosphatidylcholine levels.

UBQLN2 is a ubiquitin-binding shuttle protein that undergoes phase separation in vitro and localizes to stress-induced cellular condensates including stress granules. The central region of UBQLN2 contains two chaperone- and substrate-binding STI1 domains (STI1-I, STI1-II) and disordered linkers; the individual contributions of these domains and linkers to cellular condensate partitioning remain poorly characterized. Here we use live-cell imaging and immunofluorescence experiments to systematically examine domain requirements for UBQLN2 puncta formation in cultured human cells. We show that in vitro phase separation propensity largely correlates with puncta formation in transfected cells. Importantly, STI1-II and UBA domains are each required for baseline puncta formation in cells, but not STI1-I. In contrast, both STI1 domains are required for heat stress-induced puncta formation. Removal of STI1-II abrogates this stress response, and STI1-I deletion substantially attenuates it. Using N-terminal truncation constructs, we demonstrate that STI1-I strongly promotes both phase separation and puncta formation in the absence of the N-terminal region containing the UBL domain. Together, our findings demonstrate that the two STI1 domains of UBQLN2 have distinct roles in puncta formation and condensate partitioning, with STI1-II essential under all conditions.

The master kinases of the DNA damage response (DDR), ATR, ATM and DNA-PK, become active in response to DNA damage and orchestrate a downstream wave of phosphorylations contributing to DNA damage repair and preservation of cellular homeostasis. Of them, we recently demonstrated that ATM binds the pool of the lipid phosphatidyl-inositol-4-phosphate (PI4P) situated at the Golgi membrane. Depending on PI4P availability at Golgi membranes, ATM is more or less titrated away from the nucleus, which translates into responses to nuclear DNA damage of matching intensity. Building on this knowledge, in this work we asked if, beyond the Golgi merely serving as a docking platform that retains ATM away from the nucleus, ATM does exert any role important for Golgi biology. We found that ATM maintains Golgi morphology by counteracting its excessive deployment. This occurs both by its mere presence (likely antagonizing the Golgi-stretching action of the protein GOLPH3) and by phosphorylating Golgi-resident substrates. Of relevance, we also report that the morphological alterations caused to the Golgi without ATM affect the biology of a model Golgi cargo. Our findings nourish the growing evidence that kinases of ATMs family display functional interactions with membranes and highlights an underappreciated crosstalk between the Golgi and the nucleus.

Automating cellular organelle segmentation is key to increasing the throughput in electron microscopy (EM) and volume EM (vEM) workflows. Deep learning (DL) has accelerated this process, but model development has predominately centered on mitochondria, partly because of a scarcity of suitable training datasets for other features. Here, we crowdsourced the manual step of labeling nuclei and lipid droplets (LDs) from complex cellular EM images and trained Panoptic DeepLab (PDL) models on these large, heterogenous annotated datasets as well as on publicly available vEM datasets. NucleoNet and DropNet, the resulting instance segmentation models for nuclei and LDs, respectively, yield high-quality results on varied benchmarks. We applied these models to quantify differences between 2D and 3D in vitro cancer models versus in vivo tumors, highlighting a path toward robust quantitation in EM. NucleoNet and DropNet are publicly available on our napari plugin, empanada v1.2, for easy point-and-click segmentation of 2D and 3D cellular EM images.

Cilia are conserved microtubule-based organelles required for signaling and fluid transport, and their dysfunction causes ciliopathies. Clinical overlap between sensory and motile ciliopathies suggests that primary and motile ciliogenesis depend on shared regulatory modules. Here, we identify Centrosome and Spindle Pole-associated Protein 1 (CSPP1), a microtubule-associated protein mutated in the neurodevelopmental ciliopathy Joubert syndrome, as a conserved regulator of vertebrate multiciliogenesis. Using mouse tracheal epithelial cultures and Xenopus embryonic epidermis, we show that CSPP1 localizes to fibrous granules and deuterosomes during centriole amplification, and to basal bodies and ciliary axonemes in differentiated multiciliated cells. Loss of CSPP1 impairs centriole amplification, basal body apical migration, spacing, and rotational polarity, and is accompanied by disorganization of the apical microtubule network. CSPP1 depletion also disrupts axoneme assembly, resulting in fewer and shorter cilia with ultrastructural defects, reduced ciliary beating, and impaired cilia driven fluid flow in vivo. Together, our findings identify CSPP1 as a conserved regulator of multiciliogenesis and motile cilia function and establish a basis for future work on how shared cytoskeletal pathways may underlie overlapping features of sensory and motile ciliopathies.

Nuclear transport receptors are best known for mediating nucleocytoplasmic transport (NCT) through nuclear pore complexes. Here, we uncover an unexpected function of the primary import receptor importin-{beta} (Imp{beta}) as a direct regulator of the actin cytoskeleton. Imp{beta} associates with stress fibers and the cell cortex and promotes actin polymerization through direct interactions. Disrupting Imp{beta}-actin binding impairs stress fiber formation and suppresses cell migration well before NCT is affected. In 3D spheroids, perturbing Imp{beta} further compromises tissue integrity, as reflected in changes to nuclear curvature and ellipticity. Together, these findings identify Imp{beta} as a regulator of actin organization in both 2D and 3D contexts, revealing a direct link between the nuclear transport machinery and the cytoskeleton.

Robust chromosome segregation requires the anaphase spindle to both preserve and remodel its structure under force. How it does so remains unclear as probing mechanics during anaphases short lifetime is challenging. Here, we use microneedles to pull on mammalian anaphase midzone bundles and ask how they respond to and transmit force across space and time. We find that midzone bundles locally transmit force to each other in the spindles short axis over multiple timescales. Along the spindles long axis, midzone bundles globally transmit force: rather than bundles sliding apart or detaching under force, the spindle shortens. This reveals strong anchorage and resistance to outward sliding, and that spindle elongation requires all midzone bundles to elongate. This global force transmission is stronger for short-lived forces and is strictly PRC1-dependent, indicating limited mechanistic redundancy. In sum, the anaphase spindle acts as a single mechanical unit over short timescales to resist and transmit force, while remodeling over long timescales to segregate chromosomes. SummaryUsing microneedle manipulation, Mullin-Bernstein et al. show that microtubule bundles of the mammalian anaphase spindle are mechanically coupled, transmitting force both laterally and between spindle poles. These strong connections may help ensure coordinated and error-free chromosome segregation.

Mitochondrial-derived compartments (MDCs) are remodeling domains that form from the outer mitochondrial membrane during metabolic and proteotoxic stress and selectively sequester hydrophobic membrane proteins. Although MDC formation depends on mitochondrial lipid composition and occurs at organelle contact sites, the molecular mechanisms that permit their biogenesis remain poorly defined. Here we identify the conserved inner mitochondrial membrane i-AAA protease Yme1 as a critical regulator of MDC formation. Loss of Yme1 blocks MDC biogenesis in response to multiple stressors, and this requirement depends on its proteolytic activity rather than secondary defects in mitochondrial morphology. Quantitative mitochondrial proteomics under MDC-inducing conditions revealed Yme1-dependent remodeling of lipid transfer proteins of the Ups family and components of the MICOS complex. Disruption of either pathway partially restores MDC formation in yme1{Delta} cells, while combined perturbation substantially bypasses the requirement for Yme1. Finally, Yme1 overexpression drives MDC formation in the absence of stress, although this activity remains constrained by metabolic conditions. Together, these findings support a model in which Yme1-dependent proteolysis relieves lipid- and MICOS-dependent constraints to permit MDC formation.