搜索结果：Molecular biology of the cell

Actin bundle-supported membrane protrusions contribute to a range of physiological functions across cell types. An interesting case is the tuft cell, which assembles exaggerated microvillus-like protrusions that support chemosensory functions in the intestinal tract. Tuft cell protrusions are supported by large core actin bundles; several F-actin bundlers are expressed in tuft cells, and these exhibit regionalized localization along the core bundle axis. Uniquely, LIM domain and actin binding 1 (LIMA1) is restricted to the basal, rootlet ends of these bundles. How LIMA1 impacts protrusion formation and core bundle architecture remains unclear. Here, we leveraged multiple forms of microscopy to examine the impact of LIMA1 on the actin cytoskeleton in epithelial and nonepithelial models. Our findings indicate that LIMA1 expression is sufficient to drive the elongation of microvilli in epithelial cells, as well as the formation and stabilization of exaggerated filopodia in a non-epithelial context. Importantly, we found that individual filopodia in LIMA1-overexpressing cells exhibit merging dynamics, which enable the formation of large core bundles from smaller precursors. These data indicate that LIMA1 exerts potent effects on rootlet actin architecture and is well positioned to support the formation and maintenance of large core actin bundles, like those assembled by tuft cells.

An evolutionarily conserved E3 SUMO ligase, Mms21, orchestrates genome integrity processes. Our study examined a mutant of Saccharomyces cerevisiae Mms21, analogous to a mutant identified in a rare human condition characterized by genome instability. The human mutation C-terminally truncated the Mms21 protein, without affecting the residues in the E3 ligase domain. Thus, we hypothesized that the C-terminus regulated ligase-independent functions of Mms21. Truncating the last 22 amino acids of yeast Mms21-designated as mms21Δ22 mutants-mimicked the human disease mutation. mms21Δ22 mutants exhibited slower growth and increased DNA damage sensitivity than the wild-type and two well-characterized mutants of Mms21-one with two missense mutations in the enzymatic domain and another without the entire enzymatic domain and the C-terminus. Furthermore, mms21Δ22 mutants exhibited a G2-M delay during unchallenged growth. The mms21Δ22 allele reduced Mms21 protein levels, but the phenotypes of mms21Δ22 mutants simply could not be attributed to diminished protein levels. Our genetic data suggested that the C-terminus contributed to both ligase-dependent and -independent functions of Mms21 and opposed the activity of the adjacent domain, thereby fine-tuning genome integrity. The mms21Δ22 disease allele analogue further enhanced our understanding of Mms21's functions beyond its ligase activity in genome instability conditions.

Meiosis ensures the formation of haploid gametes through two rounds of chromosome segregation after one round of DNA replication. How this cell-cycle process is restricted to two, and only two divisions, is poorly understood. In budding yeast, RNA-binding protein Rim4 binds various mRNAs to prevent their translation. At the onset of meiosis II, phosphorylation and degradation of Rim4, along with the concomitant release of sequestered mRNA, play an important role in ensuring meiotic exit. Building on previous work, we developed a parsimonious mathematical model of meiotic termination that elucidates the role of Rim4-mRNA release and translation of AMA1 mRNA in the fidelity of meiotic exit. Central to our model is the accumulation of Ama1 protein, a meiosis-specific activator of APC/C. Our mathematical model predicted further outcomes, which we tested experimentally. We found that either slowing Rim4 degradation or disrupting APC/CAma1 activity delayed meiosis II. In some cells, this disruption prevented meiotic exit entirely, leading them to re-enter cell-cycle oscillations after meiosis II. These findings demonstrate that the timely activation of this regulatory network is crucial for ensuring irreversible meiotic exit.

Endothelial cells promote thrombosis and hemostasis through the secretion of von Willebrand Factor (VWF) from their secretory granules-the Weibel-Palade bodies (WPBs). In response to specific stimuli, dynamic actin nucleation and remodeling of the cytoskeleton facilitates the expulsion of ultra-large VWF multimers to elevate plasma VWF and to form a platform for platelet capture and thrombus formation. P21 activated kinase 2 (PAK2) is a crucial regulator of the actin cytoskeleton and is essential for VWF secretion in response to secretagogues which utilize actomyosin mediated exocytosis. Here, we characterize the role of β-PAK-interacting exchange factor (β-PIX) in WPB exocytosis. Inhibition of β-PIX function prevents dynamic cytoskeletal remodeling resulting in reduced VWF secretion. Depletion of β-PIX using siRNA reduced the number of WPB fusion events, prolonged the time taken for GFP-VWF to be secreted post-fusion and delayed kinetics of the exocytic actomyosin ring. Use of full length and truncated β-PIX demonstrated that the PAK interacting and GEF domain mediate cytoskeletal remodeling whereas only the full-length construct could rescue VWF secretion. β-PIX regulates both septin ring formation and cofilin mediated actin remodeling during actomyosin ring function. These data identify β-PIX as a regulator of endothelial exocytosis through supporting actomyosin-mediated expulsion of VWF.

Membrane tethering is essential for the generation of organelle contact sites, the catabolic process of autophagy, and to anchor incoming vesicles to their target membranes before vesicle fusion. Although membrane tethering is critical for cellular function, many of the current biochemical techniques to test for membrane tethering rely on indirect readouts and are limited in their ability to monitor protein localization at sites of tethering. As such, we recently developed a fluorescence microscopy-based giant unilamellar vesicle and liposome tethering assay (GLT) to study the membrane tethering properties of two autophagy proteins. In this study, we used GLT with engineered membrane tethers to demonstrate the ease of use, methods of analysis, versatility, and sensitivity of the assay. We demonstrate that: 1) GLT can be used to study liposome tethering, fusion, and phosphatase-mediated detethering of tethered liposomes, 2) GLT detects tethering with comparable sensitivity with less direct methods for monitoring membrane tethering while allowing simultaneous monitoring of membrane and protein localization, and 3) GLT can be used to monitor the kinetics of membrane tethering in real time. Collectively, our results demonstrate GLT is a broadly useful method to study membrane tethering in vitro.

Giardia intestinalis is a globally prevalent cause of waterborne diarrheal disease, yet about 40% of its proteome remains functionally uncharacterized due to the lack of conserved homologous proteins and limited experimental validation of protein function. To begin addressing this gap, we created a large-scale subcellular localization resource by fluorescently tagging and imaging 608 Giardia proteins (12% of the proteome) expressed in live cells from native promoters. This dataset includes 240 hypothetical proteins, 215 domain-family proteins (including ankyrin repeat and NEK kinase families), 171 diplomonad- or Giardia-specific proteins, 69 conserved eukaryotic proteins, and 77 proteins with known functions that were previously unlocalized. Imaging revealed localization to cytoskeletal and Giardia-specific organelles (eight flagella, the ventral disk, and the median body), along with novel components of the plasma membrane and endomembrane systems. Integrating localization data with domain architecture, homology, and Giardia-specific Gene Ontology terms, we produced a "localization-informed" gene annotation with a standardized, structured nomenclature. This resource provides the largest experimentally validated functional annotation of the Giardia proteome to date, linking predicted gene models to cellular structures, creating testable hypotheses for protein function and establishing a durable framework for future studies of cell biology, pathogenesis, and eukaryotic evolution in this deeply divergent diplomonad lineage.

Iron overload cardiomyopathy (IOC) is caused by elevated systemic iron, and it is characterized by systolic and diastolic dysfunction as well as arrhythmias. Isolating the cardiac-specific cellular and molecular mechanisms driving IOC has been challenging because it affects multiple interconnected organ systems. Here, we leverage stem cells, cardiac tissue engineering, and protein reconstitution to model key contractile aspects of human IOC in vitro and probe the cellular and molecular mechanisms driving cardiac dysfunction. Human-engineered heart tissues consisting of both cardiomyocytes and cardiac fibroblasts faithfully recapitulate key aspects of the human disease, including reduced contractile function, impaired relaxation, and increased prevalence of arrhythmogenic events. While both cardiomyocytes and cardiac fibroblasts show increased intracellular iron levels, cardiomyocytes show higher iron accumulation and reactive oxygen species production. Moreover, iron overload has little effect on the action potential kinetics in engineered heart tissues; however, it impacts the kinetics of the calcium transient, potentially driving arrhythmogenesis. Finally, iron overload decreases force production, in part, through oxidative damage of sarcomeric proteins and iron-based inhibition of myosin. Our results reveal insights into the cellular and molecular mechanisms of human IOC pathogenesis and establish in vitro models that can be harnessed for mechanistic and translational studies.

Cell elongation and cytokinesis are mediated by two large multi-protein complexes in rod-shaped bacteria: the elongasome and divisome. These membrane-associated complexes ultimately recruit peptidoglycan cell wall remodeling and synthesis machineries, thus sculpting and extending growing cells or driving septation of two daughter cells. The elongasome and divisome have been well studied in model bacterial species, including Bacillus subtilis, Caulobacter crescentus, and Escherichia coli. Here, we present an analysis of these complexes in an obligate intracellular bacterium with a highly reduced genome. Orientia tsutsugamushi, a cytoplasm-dwelling Gram-negative alphaproteobacterium, only retains a subset of proteins normally found in the elongasome and divisome. It also lacks all copies of the major peptidoglycan polymerase, Class A Penicillin Binding Protein, and has been shown to build an intermediate peptidoglycan cell wall-like structure that is low in abundance and insufficient to consistently confer a rod shape to the bacterium. We have carried out a first analysis of the elongasome and divisome in Orientia tsutsugamushi, quantifying the expression and subcellular localization of five key proteins through early stages of the intracellular infection cycle. We show how these are affected by antibiotic treatment and present a model for minimal elongation and division in an obligate intracellular bacterium.

The SAF-A/HNRNPU gene encodes an abundant nuclear protein conserved throughout vertebrates, and is mutated in individuals with HNRNPU syndrome, a neurological human disease. SAF-A is important for maintaining lncRNA localization, splicing, and gene expression state. The mechanistic role of SAF-A in each of these processes is likely coordinated by one or more of its functional domains, which include an N-terminal SAP domain, a central ATPase domain, and a RGG domain defined by a series of C-terminal RGG/RG repeats embedded within a low-complexity region. However, a comprehensive analysis to identify which SAF-A domains are required for each cellular function is lacking. Here we use an allelic reconstitution strategy to investigate the role of the SAF-A ATPase and RGG domains in lncRNA localization, nuclear dynamics, transcription, splicing, and cell viability. We show that both the ATPase and RGG domains control SAF-A nuclear dynamics, and that SAF-A interacts with nascent RNA Pol II transcripts through the RGG domain. The SAF-A ATPase and RGG domains were required for maintaining XIST RNA and facultative heterochromatin marks on the inactive X chromosome, but did not affect X-linked gene silencing. The SAF-A ATPase and RGG domains were both required for proper mRNA splicing, but not for gene expression. Importantly, we found that the SAF-A ATPase and RGG domains are required for cell proliferation, arguing that these domains are each linked to the essential cellular functions of SAF-A. Collectively, our findings highlight the importance of the SAF-A SAP, ATPase and RGG domains in vital functions of nuclear biology.

It has been known for over 80 years that bacterial cell size is affected by growth conditions. Cells grown in nutrient rich medium are larger and wider than those grown in nutrient poor medium. Yet even after decades of research it is still not fully known how metabolism and cell size are coregulated. In this work, we describe a new source of metabolic control over Escherichia coli cell size, the phosphoenolpyruvate phosphotransferase system (PTS). The PTS is used to phosphorylate sugars upon entry into the cell. We found that mutations in this system result in both shorter and thinner cells and that the regulation of both dimensions of cell size appears to come from two separate mechanisms. The first mechanism regulates cell length through the production of cAMP, while the second mechanism regulates cell width through control of the levels of PEP or pyruvate in the cell.

In diderm bacteria, the outer membrane (OM) must invaginate in concert with septal peptidoglycan (PG) remodeling during cytokinesis. One OM lipoprotein, DolP, has been shown to localize at the cell division site and facilitate the daughter cell separation. Yet how DolP is recruited remains unclear at the molecular level. Here, we show that DolP arrives at mid-cell concomitantly with the late divisome protein FtsN. Utilizing single-particle tracking Photoactivated Localization Microscopy (spt-PALM), we investigated the dynamics of individual DolP molecules in living Escherichia coli cells. Single-molecule analysis revealed two diffusion states: a diffusive state across the cell envelope and an immobile state enriched at the septal and polar regions. Because anionic phospholipids are known to be enriched at regions of high negative curvature, we tested mutations in the DolP anionic phospholipid-binding surface and found they abolished mid-cell enrichment and reduced the immobile fraction. Importantly, DolP's localization is independent of division proteins like EnvC and NlpD, and DolP does not comigrate with the core septal synthesis complex FtsW-FtsI-FtsN complex. Instead, DolP enrichment requires an actively constricting septum. Together, these findings support a model in which anionic phospholipid-mediated diffusion-state switching drives DolP enrichment at the division site.

Fully functional neural competence and integrity require a complex array of communication means among neurons, with extracellular vesicles (EVs) emerging as a relevant mechanism for cell-cell interaction in the CNS. Despite the growing number of studies demonstrating the presence of microRNAs (miRNAs) in axons and EVs, the molecular mechanisms of those miRNAs present in EVs and their functional role in nervous system development have not been fully explored. In this study, we investigated whether neuronal EVs can have a role in neuron-to-neuron communication during the development of neuron connectivity in mouse primary cortical neuron cultures. Our results demonstrate how miR-99a can regulate axonal growth via its EV-mediated delivery and through the targeting of HS3ST2, a heparan sulphate glucosamine 3-O-sulphotransferase, which is predominantly expressed in the brain and generates rare 3-O-sulphated domains in heparan sulphate proteoglycans, with growing importance in development and neurodegenerative mechanisms. Importantly, we show how in compartmentalized microfluidic cultures, where axons are isolated from neuronal somas, the growth-promoting effects of neuron-derived EVs are local to the axon. These findings establish that neuronal EVs can deliver miRNAs to discrete subcellular domains to acutely modulate local gene expression, thereby driving axonal growth and shaping neurodevelopment.

Despite the success of long-term antiretroviral therapy (ART), immune dysfunction-including incomplete T helper cell recovery, immune activation, and inflammation-persists and may affect the benefits of analytical ART treatment interruption (ATI) trials among people living with human immunodeficiency virus (HIV) (PLHIV) in sub-Saharan Africa (SSA). This narrative review summarizes evidence of incomplete immune function recovery among PLHIV on long-term ART and potential interventions to enhance their ability to participate in ATI trials with immunotherapies in the quest for an HIV cure in SSA. A PubMed search query was used "([Immunity, Innate{MeSH Terms}] OR [Adaptive Immunity{MeSH Terms}] AND [HIV{MeSH Terms}] AND [Anti-HIV Agents{MeSH Terms}] AND [function{Title/Abstract} OR dysfunction{Title/Abstract} OR recovery{Title/Abstract} OR restoration{Title/Abstract} OR reconstitution{Title/Abstract} OR regeneration{Title/Abstract}])", which retrieved 165 articles. These articles were filtered using an English-language filter, resulting in 160 papers. This query was translated to Web of Science and Google Scholar. In addition, we conducted a specific literature search on documented "cured" HIV cases globally to understand innate and adaptive immune functions relevant to supporting post-ART immunological viral control. Persistent dysfunction of host innate and adaptive immunity during suppressive ART is reported in SSA HIV treatment cohorts. Natural killer (NK) cells, dendritic cells, and monocyte dysfunction potentially limit post-ART viral control. In addition, persistently impaired CD4 and CD8 T-cell proliferation capacity, immune activation, and exhaustion during ART may limit host HIV-specific responses during ATI trials with broadly neutralizing antibodies (bNAbs) and therapeutic vaccines, thereby increasing the risk of post-ART viral rebound. Similarly, adjunct therapies with TLR7 agonists, such as vesatolimod, could potentially increase cytotoxic capabilities of dendritic and natural killer cells to improve HIV latency reversal and viral clearance during ART. Innovative combination immunotherapies to avert persistent immune dysfunction, such as therapeutic vaccines, combination bNabs, enhancer of zeste homolog 2 (EZH2) inhibitors to boost CD8+ T-cell function and augment post-ART viral control, and latency reversal agents to increase cytotoxicity potential of dendritic cell and natural killer cells, among others, could potentially optimize HIV-specific CD8 T-cell cytotoxicity and viral clearance, and delay viral rebound during ATI trials in SSA.

Epithelial wound healing is an essential process in multicellular organisms, primarily driven by lamellipodia-based crawling, purse-string contraction, and collective cell migration. One or more of these mechanisms participate in healing a wound, yet the choice, sequence, and coordination of these processes are poorly understood. Moreover, different mechanisms dominate in different tissues, organisms, wound types, and wound sizes, further complicating our understanding of how cells select among healing mechanisms. In this study, we analyzed wound healing across wound types and spatial scales in the basal eukaryote Clytia hemisphaerica (Clytia) to establish a unified model for mechanism selection within a single organism. We demonstrate that lamellipodial crawling and actomyosin cable contractions are sequential, partially redundant processes involved in healing all wounds. Furthermore, the exposure of the basement membrane acts as a central regulatory cue, orchestrating lamellipodia formation, actomyosin contraction, and collective cell migration responses. Remarkably, we discovered that these same mechanisms operate in healing micro-wounds internal to a single cell. This work fundamentally advances our understanding of how diverse healing mechanisms are coordinated to respond to all types of wounds, while the use of a basal metazoan model expands our knowledge of fundamental strategies for maintaining epithelial integrity.

Yeast cells rely on the actomyosin machinery to mediate organelle motility. The type V myosin motor Myo2p transports organelles along actin cables from the mother cell to the nascent bud. To facilitate this process, organelles have evolved specific adaptor proteins that link them to the cargo-binding domain of Myo2p. Peroxisomes use two such adaptors: Inp2p, the principal determinant of peroxisome inheritance, and the biogenesis factor Pex19p, which has been assigned a secondary role in peroxisome partitioning. Here, we identify a regulatory function for the peroxisome biogenesis factor Pex3p in controlling peroxisome inheritance in the budding yeast, Saccharomyces cerevisiae. Pex3p is an integral membrane protein that contains a cytosol-exposed surface loop subject to phosphorylation. This loop is essential for the recruitment of both Inp2p and Pex19p. Phosphorylation of serine residues within the loop in response to environmental stimuli abolishes Inp2p binding to, and markedly reduces Pex19p association with, Pex3p, thereby reducing the efficiency of peroxisome partitioning between the mother cell and the bud. Deleting the loop in Pex3p completely abolishes peroxisome segregation. By serving as a membrane anchor for the recruitment of both inheritance factors Inp2p and Pex19p, Pex3p exerts a critical level of control over peroxisome partitioning.

The Anaphase-Promoting Complex/Cyclosome (APC/C) is a ubiquitin ligase that promotes the ubiquitination and subsequent degradation of numerous cell cycle regulators during mitosis and in G1. Proteins are recruited to the APC/C by activator proteins such as Cdh1. During the cell cycle, Cdh1 is subject to precise regulation so that substrates are not degraded prematurely. We have explored the regulation of Cdh1 during the developmental transition into meiosis and sporulation in the budding yeast Saccharomyces cerevisiae. Transition to sporulation medium triggers the degradation of Cdh1. Cdh1 degradation is mediated by the APC/C itself in a "trans" mechanism in which one molecule of Cdh1 recruits a second molecule of Cdh1 to the APC/C for ubiquitination. Degradation requires an intact glucose-sensing SNF1 protein kinase complex (orthologous to the mammalian AMPK nutritional sensor), which directly phosphorylates Cdh1 on Ser-200 within an unstructured N-terminal region. In the absence of phosphorylation, expression of a Cdh1-S200A mutant is fully stabilized, leading to defective meiosis or spore formation and loss of viability. We hypothesize that Cdh1 degradation is necessary for the preservation of cell cycle regulators and chromosome cohesion proteins between the reductional and equational meiotic divisions, which occur without the intervening Gap or S phases found in mitotic cell cycles.

Cytokinetic abscission genes are linked to cancers and developmental disorders, but the consequences of disrupted abscission in vivo remain under-explored. Previously we showed that in the forebrain of Cep55 knockout (KO) mouse embryos, a subset of neuroepithelial stem cells (NSCs) fail abscission and become binucleate, and some of those undergo p53-mediated apoptosis. Here we use the Cep55 KO to investigate how stochastic abscission failures in a polarized epithelium affect the epithelial architecture. We find that NSCs in Cep55 KO neuroepithelium have preserved epithelial polarity and integrity. However, they have enlarged apical membranes (called apical endfeet), longer primary cilia, and increased biciliation. We then test whether the enlarged apical endfeet arise from filling the space of apoptotic neighbors. Remarkably, blocking apoptosis does not rescue but exacerbates the phenotypes: extra-large apical endfeet have further increased multiciliation, supernumerary centrosomes, and abnormal or multiple nuclei, although epithelial polarity is maintained. These findings elucidate the importance of proper abscission in maintaining polarized epithelial structure, and reveal that p53-mediated apoptosis is a crucial guardian of tissue architecture when cell division defects arise during development and disease.

Biomolecular condensates are central to subcellular compartmentalization and RNA regulation. In the multinucleate fungus Ashbya gossypii, condensates composed of Whi3 protein and CLN3 mRNA help ensure nuclear cycle asynchrony in a shared cytoplasm. Here, we investigated how Whi3 protein binding sites within CLN3 mRNA are specified and influence properties of the condensate. We found that Whi3 binds to varied RNA sequences but prefers the five-nucleotide motif UGCAU, which appears at five locations in the CLN3 transcript. Mutating individual UGCAU motifs altered the saturation concentration (Csat) and dense phase concentration of RNA and Whi3 in cell-free reconstitution experiments. These defects were partially rescued by melting and refolding the mRNA, indicating that RNA structure plays a critical role in distinguishing binding sites and determining condensate properties. Lastly, a subset of mutants showed reduced condensate numbers and dysregulation of the cell cycle in cells. These data reveal that the context of otherwise identical mRNA sequences can differentially affect condensate properties.

Expression of late G1 phase cyclins is the critical molecular event that marks commitment to enter the cell cycle. In budding yeast, late G1 phase cyclins initiate and sustain the growth of a new daughter bud, so their expression also marks the start of a new growth phase during the cell cycle. Expression of late G1 phase cyclins is influenced by nutrient availability-cells growing in poor nutrients progress through the late G1 phase with lower levels of late G1 phase cyclins. However, little is known about how or why nutrients modulate expression of late G1 phase cyclins. Here, we investigated the signals that control expression of the late G1 phase cyclin Cln2. We discovered that nutrients modulate the expression of Cln2 via post-transcriptional mechanisms that influence Cln2 phosphorylation and turnover. Nutrient modulation of Cln2 protein expression requires a TORC2-MAP kinase signaling axis. Expression of Cln2 is closely correlated with bud growth and is required for bud growth. A model that could explain the data is that nutrients modulate Cln2 expression to ensure that the rate of bud growth is matched to the availability of nutrients that support bud growth.