Cell Structure and Function

The small GTPase Rac1 controls cell protrusion for a wide variety of critical cell functions. Its regulation by upstream guanine exchange factors (GEFs) has been the focus of multiple studies, but regulation by the GTPase RhoG remains poorly understood. RhoG is known to activate the ELMO/DOCK180 GEF complex, which in turn interacts with Rac1. It is unclear which aspects of protrusion are controlled by RhoG, and which of RhoGs effects on protrusion are mediated by Rac1. To address these questions, we developed biosensors and optogenetic tools to activate one GTPase while observing another, and to simultaneously visualize the activity of two GTPases. New tools included a photoactivable RhoG, a RhoG biosensor, and red shifted biosensors of RhoG and Rac1. RhoG and Rac1 activation events in protrusions were spatio-temporally correlated with one another and with protrusion velocity. Causal inference indicated that RhoG indeed unidirectionally activated Rac1. Photoactivation of RhoG and Rac1 indicated that specific aspects of protrusion behavior were controlled by RhoG, and only some via Rac1. Further dissection of RhoG to Rac1 signaling through simultaneous GTPase activation and biosensor visualization showed that PA-RhoG activates Rac1 predominantly through DOCK180 and that PA-RhoG can activate Cdc42 independently of Rac1.

Copines are a family of calcium-dependent phospholipid-binding proteins found in most eukaryotes. The expression of multiple copine genes is dysregulated in various types of human cancers. Despite this, a common mechanistic function for copines remains unknown. We are studying copines in Dictyostelium discoideum, which has six copine genes (cpnA-cpnF). Cells lacking cpnA or cpnC (cpnA- and cpnC-, respectively) exhibit many phenotypes, including defects in development, chemotaxis, adhesion, and contractile vacuole (CV) function. To further characterize the function of copines, this study tested the hypothesis that CpnD is responsible for cellular functions distinct from CpnA and CpnC. In this study, we obtained two cpnD mutants that were generated via restriction enzyme-mediated integration (REMI) mutagenesis; one in the first exon (cpnD(i291)), and one in the second exon (cpnD(i459)) of the endogenous cpnD gene. Throughout our experiments, we found that cpnD mutants had increased cellular proliferation in both axenic and bacterial cultures. Additionally, we found that cpnD mutants exhibited precocious development and had significantly larger fruiting bodies than the parental cell line. We further investigated the morphology of cpnD mutants and found that they were significantly larger than parental cells and exhibited decreased cell-substrate adhesion. cpnD mutants also had increased activated Ras compared to the parental cell line, along with significantly smaller CVs, a phenotype that was rescued after PI3K inhibition. Finally, we found that GFP-tagged CpnD localizes to the leading edge of both randomly migrating cells and in cells responding to folate. This study is the first to describe copine proteins as having a regulatory function in Ras activation and downstream signaling effects. Additionally, this study further supports our hypothesis that copines act as nonredundant cellular proteins in Dictyostelium to regulate numerous processes.

Though whole-genome duplication (WGD) contributes to cancer progression, the mechanism of post-WGD cell proliferation remains unclear. Here, using 6-day live-imaging, we analyzed the proliferation dynamics of more than 150 post-WGD HCT116 cell lineages. A quantitative comparison of mitotic patterns and cell fates between proliferative and non-proliferative lineages revealed that multipolar chromosome segregation in early mitosis is a key factor limiting the proliferative capacity of post-WGD progenies. Multipolar chromosome segregation suppressed post-WGD cell viability, particularly when accompanied by drastic chromosome loss or when it repeatedly occurred. Tracing proliferative lineages elucidated that they proliferated mainly by imposing the risk of multipolar chromosome segregation on one of two sub-lineages that formed after the first bipolar division. Meanwhile, a considerable proportion of proliferative lineages consisted entirely of progeny of early multipolar chromosome segregation events. Our results highlight key cellular events that determine the proliferation dynamics and diversity of post-WGD progenies, providing a fundamental reference for understanding WGD-associated bioprocesses. Summary statementLive image tracing of >150 cell lineages reveals the cross-generation dynamics of multipolar chromosome segregation that determine the fates of post-whole-genome duplication progeny cells.

Tumor heterogeneity highlights the necessity of precision cancer medicine, making the evaluation and screening of anticancer drugs a core challenge in cancer therapy. However, current cell-based efficacy assessment methods struggle to quantify the holistic impact of drugs on cellular behavior through specific target engagement. Here, we proposed a novel approach (DL-TCP-FRET) that integrates phenotypic and target-related evaluations: the logistic fitting analysis is performed on time- and concentration-dependent cellular phenotypic characteristics to construct a phenotypic score (P), while a target score (T) is established based on the FRET efficiency between target proteins. These two scores were then further combined to generate a unified drug efficacy score (PT). Validation in A549 cells demonstrated that our method can reliably distinguish EGFR-TKIs from non-targeted drugs. DL-TCP-FRET simplifies the experimental workflow of drug efficacy evaluation and improves the accuracy of targeted drug identification, providing a novel strategy for advancing precision cancer therapy.

Nucleoli and centromeres play essential roles in cellular proliferation and homeostasis, and are structurally and functionally interconnected. Centromeres frequently cluster around nucleoli, and some centromere assembly factors are known to reside in the nucleoli. To investigate the spatial and temporal relationships between these nuclear domains, we examined their dynamics in living cells. We imaged HeLa cells stably expressing mCherry-NPM1 and GFP-CENP-A using time-lapse microscopy. The results show that a subset of centromeres exhibits dynamic behavior during interphase, migrating over micrometer-scale distances within two hours. On average, 40-50% of centromeres maintain an association with nucleoli throughout interphase, with some cells displaying nucleolar-centromere association and dissociation within hours. Upon entry into mitosis, nucleoli are disassembled, and NPM1 localizes to the periphery of mitotic chromosomes. Nucleolar-centromere interactions are re-established in early G1, coinciding with the assembly of new centromeres. Treatment with actinomycin D, an inhibitor of RNA polymerase I, significantly reduces nucleolar size, nucleolar-centromere interactions, and centromere dynamics. Furthermore, post-mitotic nucleolar reformation is impaired. These findings highlight the dynamic nature of centromeres in interphase nuclei and their interactions with nucleoli. This behavior is partially dependent on rDNA transcription and nucleolar integrity, underscoring the critical roles of nucleoli, centromeres, and their interaction in 4D genome organization.

Multicellular organisms comprise various types of cells, which are characterized by gene expression through interactions between chromosomal DNA and nuclear proteins. Many cutting-edge methods have been developed to reveal the three-dimensional organization of chromosomes. The detailed analyses of whole chromosomes have begun to uncover structural features specific to several cell types. Here, we show that cell types are instantly and highly accurately classified using conventional DNA staining and a convolutional neural network (CNN). A high-resolution single slice image of the nucleus is sufficient for the accurate classification of both live and fixed cells, including neurons and non-neural cells. These findings suggest that there may be cell-type-specific features decipherable by deep learning in a thin two-dimensional slice of the nucleus.

Cell polarity, essential for cell development and function, relies on dynamic subcellular distribution of structural and signaling molecules. Tip growth, an extreme form of polar growth, involves unidirectional expansion at the apical region of cells and requires precise spatiotemporal coordination to achieve periodic and directional growth. Understanding their spatiotemporal dynamics is critical for elucidating mechanisms and functions of cell polarity. However, manual quantification of such dynamics is extremely time-consuming, hindering advancements in the field. Current algorithms have limited power and flexibility in analyzing the distribution and dynamics of molecules and structures, particularly for tip-growing cells with oscillatory and dynamic behavior. To address this challenge, we present TipQuant, an automated analysis tool that robustly detects tips and analyzes spatiotemporal dynamics of fluorescently labeled molecules/structures on plasma membranes and in cytoplasm at apices of tip-growing cells, enabling quantitative understanding of signaling and structural components in these systems.

During maturation of a Golgi cisterna, multiple vesicular transport pathways recycle resident Golgi proteins. Recycling vesicles are captured by Golgi-associated tethers. To assign individual tethers to specific recycling pathways in Saccharomyces cerevisiae, we examined tether arrival and departure using kinetic mapping, and we examined tether function using an ectopic tether localization assay. Those approaches yielded mutually consistent results. Our analysis focused on two coiled coil golgin tethers and the multi-subunit tether GARP. At an intermediate stage of cisternal maturation, the golgin Sgm1 tethers proteins that follow an intra-Golgi recycling pathway dependent on COPI. At a late stage of cisternal maturation, GARP and the golgin Imh1 tether trans- Golgi network (TGN) proteins that follow an intra-Golgi recycling pathway dependent on the AP-1 and Ent5 clathrin adaptors. This involvement of GARP in intra-Golgi recycling had not previously been documented. Imh1 also tethers proteins that recycle from prevacuolar endosome compartments to the TGN. Our findings contribute to an integrated model of Golgi membrane traffic.

Acquiring nutrients is a fundamental biological process of all organisms, playing crucial roles in ecological sustainability. Diplonemids are highly abundant heterotrophic unicellular flagellates that are widespread in the worlds ocean. They have a highly complex microtubule-based feeding apparatus (cytostome-cytopharynx complex) located adjacent to the deep flagellar pocket from which two flagella emerge from parallel basal bodies. The apical papilla is a tongue-shaped structure unique to diplonemids that connects the cytopharynx and the flagellar pocket, the latter of which is formed by reinforcing microtubules (MTR) and two flagellar roots called intermediate and dorsal roots. Here we report identification of 17 proteins that localize at the feeding apparatus or flagellar apparatus in Diplonema papillatum. Using ultrastructure expansion microscopy, we show that Mad2 and its interaction partner MBP65 localize at the MTR, intermediate root, and dorsal root. Homologs of proteins that associate with the flagellar apparatus in Trypanosoma brucei (PFR2, KMP11, BILBO1) localize at the feeding apparatus in D. papillatum. We also identify proteins that localize at the apical papilla, MTR, parallel microtubule loop, or cytopharynx. By discovering components of the feeding apparatus for the first time in diplonemids, this work forms the foundation to understand molecular mechanisms of the feeding apparatus in these highly abundant marine plankton.

The cell cycle comprises different phases and is a tightly regulated process at the molecular level. During the cell cycle, two key events occurred: DNA duplication during the S phase and chromosome segregation during mitosis. Accurate cell cycle progression, achieved through faithful chromosome segregation, is essential for maintaining cell fidelity. Long noncoding RNAs are a subclass of noncoding RNA that are longer than 200 bp and form RNA protein complexes (RNPs) to regulate various biological processes. Herein, we demonstrate that lncRNA NORM is involved in regulating the cell cycle by maintaining proper chromosome segregation. NORM exhibited G2 phase-specific expression, and the depletion of NORM resulted in a significant G2/M arrest. NORM-depleted cells failed to progress in mitosis and showed defects in chromosome segregation. We further demonstrated that NORM binds to proteins such as Plk1 and Nsun2. Depletion of NORM hindered the interaction between Plk1 and Bub1, resulting in reduced kinetochore localization of Plk1 during prometaphase. Our results also show that the depletion of NORM affects the binding of Nsun2 protein to CDK1 mRNA and, consequently, the stabilization of CDK1 at the protein level. Altogether, our results demonstrate that NORM regulates chromosome segregation by mediating the interaction between Plk1 and Bub1.

Centrosome dysfunction is linked to developmental disorders affecting brain and body size, including microcephaly and primordial dwarfism. However, the cellular mechanisms underlying these rare conditions remain poorly understood. In this study, we investigate a rare variant of the centrosome-associated protein Pericentrin, which was discovered in a single family with Majewski/microcephalic osteodysplastic primordial dwarfism type II (MOPD II). Unlike the majority of pathogenic PCNT variants that cause severe protein truncation, the p.Lys3154del variant ({Delta}K3154) involves a single amino acid deletion in the proteins only conserved functional domain, providing a unique opportunity to explore PCNT function in MOPD II. To model PCNT{Delta}K3154, we examined the effects of Drosophila Pericentrin-like protein (PLP) carrying an orthologous deletion (Plp{Delta}R). Our results show that plp{Delta}R animals exhibit smaller tissues that recapitulate MOPD II phenotypes. Behavioral assays revealed defects in climbing and mechanosensation, suggesting impaired sensory cilia function. We also found that Plp{Delta}R cells exhibit accelerated mitosis, increased apoptosis, and reduced pericentriolar material recruitment. In silico structural modeling, yeast two-hybrid, and co-immunoprecipitation experiments show that Plp{Delta}R produces a protein that disrupts PLP dimerization and PLP interaction with Asterless, another centrosome protein. Overall, modeling the human MOPD II patient variant PCNT{Delta}K3154 in Drosophila reveals how a single amino acid deletion affects biological processes from the molecular level to the organismal level. Our work offers new insights into the defective cellular mechanisms underlying MOPD II in patients with the PCNT{Delta}K3154 variant, potentially linking the etiology of the disease in these individuals to the loss of a single protein-protein interaction.

Multiplex staining is a technique that allows the identification of cell types within a single tissue section by simultaneously detecting multiple molecular markers. Generally, multiplex staining is performed using several combinations of probes, including specific antibodies, nucleic acid probes, and lectins. Here, a novel multiplex staining strategy that relies exclusively on lectin probes that target glycans is presented. Glycans have a vast variety of structural forms that vary depending on cell type-specific modifications. Furthermore, an enormous number of glycan-binding molecules, collectively known as lectins, exist in the biological world. Each lectin displays specificity for a particular glycan motif while maintaining broad affinity. Although lectin-based cell staining has been used in various applications, the partial and limited specificity of lectins has hindered the use of glycan-targeted multiplex staining with lectins. In addition, lectin probes have largely been avoided for cell-type identification because of the absence of strict cell-type-specific glycans. Here, a novel staining method, Glycan Painting, is introduced. Rather than viewing the partial specificity of lectins and the broad, non-cell-type-specific distribution of glycans as drawbacks, this approach turns these features into advantages by generating distinct color patterns that comprehensively visualize cell-type-specific glycan combinations and enable full-color imaging of tissues.

Subcellular architecture is tightly controlled and contributes to the maintenance of cells homeostasis. Organelles are regulated in size, shape, number and position which respond to changes in extracellular environment. Lysosomes are of particular interest as they integrate various functions in the cells (nutrient sensing, metabolism, cell migration and adhesion), serving as signaling hubs. Their function is tightly linked to their subcellular position and deregulation of lysosome homeostasis leads to several diseases including cancer. Therefore, methods allowing precise analysis of organelle subcellular distribution can aid in fundamental, diagnostic and therapeutic approaches. Here, we provide a versatile image analysis pipeline using ImageJ and CellProfiler. This workflow allows to quantify subcellular lysosome distribution in living and fixed melanoma cells, and is applicable to other subcellular compartments and to various cell types.

Cell contractility plays numerous essential roles in a healthy organism, while its malfunctioning can lead to disease. The ubiquitous actin-dependent motors of the nonmuscle myosin 2 (NM2) family, which includes NM2A and NM2B, are chiefly responsible for cell contractility because of their ability to polymerize into bipolar filaments. Polymerization/depolymerization of NM2 filaments allows cells to quickly reorganize their contractile system according to constantly changing shapes and positions of nonmuscle cells. Bipolar filament depolymerization is known to depend on the C-terminal features of the NM2A heavy chain. Here, we show that the motor activity of NM2A is another key component of NM2As depolymerization mechanism, which cooperates with tail-dependent mechanisms to facilitate NM2A turnover in cells and, through copolymerization with NM2B, to reorganize and dynamize NM2B in trans, thus generating a proper intracellular NM2A/NM2B distribution needed for efficient cell migration. Together, we show that NM2A motor activity is a key component of the bipolar filament depolymerization mechanism.

Membrane contact sites (MCSs) enable communication between organelles and play central roles in lipid metabolism. In budding yeast, the nucleus-vacuole junction (NVJ) functions as a dynamic platform that integrates lipid metabolism and stress responses. However, it remains unclear whether NVJ structure and function are broadly conserved across eukaryotes, particularly because Nvj1, the key membrane tethering factor that mediates NVJ formation in budding yeast, is absent in higher eukaryotes. Here, we investigated whether an MCS analogous to the NVJ in budding yeast exists in fission yeast (Schizosaccharomyces pombe), which lacks Nvj1. We show that an NVJ is present in fission yeast and serves as a platform for the accumulation of sterol synthesis factors, including the HMG-CoA reductase Hmg1 and the INSIG homolog Ins1. We further demonstrate that the localization of these factors depends on the membrane protein insertase Snd302 and is dynamically regulated by nutrient conditions. Our findings reveal that, despite the absence of Nvj1, the NVJ is functionally conserved as a site for sterol synthesis in fission yeast, suggesting a conserved role of spatial organization in lipid metabolism.

LRRK2, the Parkinsons disease-associated kinase, phosphorylates a subset of Rab GTPases and regulates membrane dynamics. We previously reported that lysosomal stress activates LRRK2 and thereby induces the exocytic secretion of lysosomal contents, but the detailed secretion mechanism remained unclear. Here we found that, under lysosomal stress, endolysosomal luminal and membrane components were secreted with extracellular vesicles (EVs) via LRRK2. Bis(monoacylglycerol)phosphate, an endolysosomal lipid and a urinary marker of LRRK2 activity, was similarly secreted via LRRK2, whereas CD9-positive EVs were not involved. Further dissection of the secreted EVs revealed that Alix-positive EVs were secreted via Rab8a as well as the ESCRT component VPS4, whereas LAMP1/cathepsin B-positive EVs were secreted via Rab10/Rab35, and SNARE proteins syntaxin 2 and VAMP8 regulated the secretion of both EV subtypes. These findings suggest a distinctive stress-induced secretory mechanism whereby LRRK2 facilitates the secretion of multiple EV subtypes by controlling Rab GTPases involved in each pathway.

Fast cytoplasmic streaming enables extensive chloroplast movements in the giant cells of unicellular, siphonous macroalgae. Here, we studied chloroplast movements in two such algae: the Dasycladalean Acetabularia acetabulum and the Bryopsidales Bryopsis sp.. We hypothesised that chloroplast movements function as a protective avoidance mechanism under excess light, particularly in Bryopsis sp., which lacks capacity for fast induction of photoprotective non-photochemical quenching (NPQ) and state transitions. In addition, we also investigated whether chloroplast movements are involved in responses to wounding and herbivory. The movements were studied by light microscopy, photography and pulse modulated chlorophyll a fluorescence quenching analysis. Chemical inhibitors of actin polymerization and microtubules assembly were used to confirm that the observed effects were active responses controlled by the cytoskeleton. A. acetabulum responded to high light by reversible chloroplast aggregation, probed by macro-imaging; and chemical inhibition of chloroplast movements led to an enhancement of Photosystem II photoinhibition, as probed by the fluorescence parameter FV/FM. No chloroplast movements were observed in Bryopsis sp. in response to high light. In A. acetabulum, wounding caused either by cutting or due to feeding by the sap-sucking sea slug Elysia timida triggered aggregation of chloroplasts within minutes of incurring the damage. Interestingly, the aggregation also occurred in intact cells away from the cutting site. Furthermore, the addition of media collected from the vicinity of cut algae was sufficient to induce chloroplast aggregation in intact algae, suggesting that water-borne cues or signals triggered the aggregation response in A. acetabulum. Bryopsis sp., however, responded to cutting by only local chloroplast aggregation. The relevance of chloroplast movements in protection against both abiotic and biotic stressors in A. acetabulum, and the potential reasons behind the different defence strategies of the algae, are discussed.

The secretion of glucagon from the pancreatic alpha () cell within the islets of Langerhans is physiologically regulated by nutrients (glucose, amino acids, fatty acids), neurotransmitters, and paracrine hormones. Insulin and somatostatin form an intra-islet paracrine network to control glucagon secretion through direct inhibitory effects on cell secretory granule exocytosis. In a potential new cellular pathway for the regulation of glucagon secretion, we have previously identified the neuronal trafficking protein Stathmin-2 (Stmn2) as a negative regulator of glucagon trafficking and secretion by directing glucagon to degradative lysosomes. In this study, we examined if insulin and somatostatin direct glucagon to lysosomes in a Stmn2-dependent manner as part of their paracrine mechanisms. Using the TC1-6 glucagon-secreting cell line and confocal microscopy of both fixed and live cells, we show that insulin and somatostatin direct glucagon, glucagon+LAMP1+ vesicles, and LAMP1-RFP to the intracellular region, away from sites of exocytosis. As visualized in live cells, insulin treatment resulted in the rapid retrograde transport of lysosomes from the cell periphery, and this effect was lost under siRNA-mediated silencing of Stmn2. Somatostatin appeared to enhance the intracellular retention of lysosomes, also in a Stmn2-dependent manner. We determined a possible mechanism for Stmn2 in the regulation of lysosome transport in TC1-6 cells through the Arf-like small GTPase Arl8, indicating that Stmn2 may function in lysosomal positioning along microtubules. We propose that Stmn2-mediated lysosomal transport may be a potential new pathway, in addition to inhibition of secretory granule exocytosis, through which insulin and somatostatin regulate glucagon secretion.

Cancer cells exposed to nutrient deprivation activate adaptive programs to survive metabolic stress, often acquiring enhanced plasticity and motility. We have previously reported that colon cancer cell lines that survived nutrient depletion underwent partial epithelial-mesenchymal transition (pEMT), which was further exacerbated when these cells also underwent lysosomal alkalinization. Here, we have attempted to dissect the molecular mechanisms that drive the motility and shape change from cobblestone to elongated in subpopulations of cells. Using RNA-seq-based bioinformatic analyses integrated with pathway scoring, protein-protein interaction networks, probabilistic modeling and confirmatory experimental data, we have identified the coordinated activation of sublethal apoptotic signaling, fatty acid oxidation, mitochondrial ROS generation, and Ca{superscript 2}-dependent lysosomal exocytosis in the nutrient-depleted cells. Among these phenotypes, the cells undergoing starvation and lysosomal alkalinization exclusively mediated lysosomal exocytosis and cell motility. Probabilistic modeling further revealed non-linear relationships between metabolic stress signals and cell fate transitions, highlighting heterogeneous lysosomal functions as a key determinant of the altered phenotype of cells under nutrient depletion. Overall, our study has identified that aberrant lysosomal functioning in cells under nutrient depletion can specifically select for a subpopulation of cells that are highly viable, metabolically plastic and capable of motility.

Targeted protein degradation using conditional degron tag (CDT) technology is a powerful method for rapidly degrading a protein of interest (POI) upon the addition of a degrader drug. A prerequisite for the temporally controlled degradation of an endogenous POI is the generation of homozygous knock-in cells with the degron tag integrated at either the N- or C-terminus of their gene loci. However, obtaining those homozygous knock-in cells often requires selecting many single-cell clones, as human cells typically exhibit low homology-directed repair (HDR) activities. Additionally, tagging a degron to an endogenous protein may inadvertently reduce protein expression, potentially affecting protein function even before the drug is administered. Here, we develop a method for generating degron-tagged knock-in cells that allows us to skip the laborious single-cell cloning. This method arose from our observation that most knock-in cells carry the degron tag only in one allele (heterozygous), while the other allele typically harbors a frameshift insertion/deletion. This observation allowed us to bypass the need for single-cell cloning. We validated our method by knocking in degron tags at the N-terminus of cytoplasmic dynein1 subunits or Adaptor Protein 2 (AP2) subunit. Our experiments confirmed the rapid degradation of these proteins and their functional inhibition in bulk cell populations. Additionally, to mitigate the reduced expression often associated with the degron tagging, we established a method to control expression levels by inserting a mini-promoter immediately upstream of the knock-in cassette. Our method simplifies the workflow for degron tag knock-ins and enhances the versatility of these valuable technologies.