Journal of cell science最新文献

Vesicle-associated membrane protein-associated protein A (VAPA) is a protein of the endoplasmic reticulum (ER) and a component of several membrane contact sites (MCSs). We show here that VAPA also localizes to the inner nuclear membrane (INM), in close proximity to nuclear lamins, INM proteins and nucleoporins. Using our proteomics approach 'rapamycin- and APEX-dependent identification of proteins by SILAC' (RAPIDS), we identified several nuclear proximity partners of VAPA, including emerin, different LAP2 isoforms, lamin A/C and Nup153. Depletion of VAPA in various cellular systems resulted in reduced nuclear lamin levels and aberrant nuclear morphology, including the formation of membrane invaginations and tunnels. Furthermore, histone acetylation levels were altered. Our data suggest that VAPA has distinct nuclear functions, in addition to its established role as an ER organizer.

LAMP1 and LAMP2A are abundant proteins of late endosomal/lysosomal compartments that are often used interchangeably to label what is assumed to be the same organelle population, potentially obscuring distinct physiological roles. Here, we characterised the axonal transport dynamics of LAMP1- and LAMP2A-positive compartments in human iPSC-derived cortical neurons. We found that LAMP1-positive organelles move slower in the retrograde direction, pause more frequently, and display a broader anterograde velocity distribution than LAMP2A-positive vesicles, indicating distinct trafficking behaviours. Co-transport analysis revealed that approximately 65% of motile LAMP-positive organelles carry both markers, with higher co-transport in the retrograde direction. To explore molecular differences underlying these behaviours, we performed proximity labelling using full-length LAMP1 or LAMP2A fused to the light-activated biotin ligase, LOV-Turbo. This approach revealed largely overlapping interactomes, with LAMP2A-associated proteins forming a subset of the LAMP1 interactome and showing an enrichment for synaptic vesicle-related proteins. We further validated ZFYVE16 as a novel interactor of both compartments. Together, our findings indicate that LAMP1- and LAMP2A- positive organelles share overlapping molecular identities but represent functionally distinct axonal populations with divergent transport dynamics.

Complex transcriptional programs and signaling pathways control early haematopoietic lineage specification. Many key regulators have been identified; however, a substantial portion of the genome remains functionally uncharacterized. Here, we investigated six uncharacterized 'Riken' genes identified through transcriptomic profiling of Flk-1+/Pdgfrα- (haematoendothelial-enriched) and Flk-1+/Pdgfrα+ (cardiac mesoderm-enriched) populations at day 4 of embryoid body (EB) differentiation. We generated knockouts in mouse embryonic stem cells and performed bulk RNA-sequencing at day 4. Three of these genes (C130074G19Rik, I830077J02Rik, A530016L24Rik) were selected for further investigation by single-cell RNA-sequencing at day 7 of differentiation, which provided novel insight for two of these genes. Knockout of C130074G19Rik (G19Rik) increased the abundance of megakaryocyte progenitors and reduced endothelial populations, with differentially expressed genes enriched for hemostasis and membrane trafficking pathways. The I830077J02Rik (J02Rik) knockout cells showed subtle changes in extracellular matrix and cell adhesion genes, with a shift toward haematoendothelial lineages. Both G19Rik and J02Rik genes encode (predicted) transmembrane proteins that modulate membrane-associated processes in early haematopoietic development. This work establishes a framework for the study of uncharacterized genes with potential roles in cell fate determination.

In mitosis the duplicated genome is aligned and accurately segregated between daughter nuclei. CTCF is a chromatin looping protein that localizes to the centromere in mitosis with an unknown role. We previously published data showing that CTCF constitutive knockdown causes mitotic failure, but the mechanism remains unknown. To determine the role of CTCF in mitosis, we used a CRISPR CTCF auxin inducible degron cell line for rapid degradation. CTCF degradation for 3 days resulted in increased failure of mitosis and decreased circularity in post-mitotic nuclei. Upon CTCF degradation, CENP-E is still recruited to the kinetochore and there is a low incidence of polar chromosomes that occur upon CENP-E inhibition. Instead, immunofluorescence imaging of mitotic spindles reveals that CTCF degradation causes increased intercentromere distances and a wider and more disorganized metaphase plate, a disruption of key functions of the centromere. These results are similar to partial loss of cohesin, an established component of the centromere. Thus, we reveal that CTCF is a key maintenance factor of centromere function, successful mitosis, and post-mitotic nuclear shape.

Tip growth is closely tied to fungal pathogenicity. Budding yeast Spa2 (the homolog of GIT1 and GIT2 in mammals), a multi-domain protein and member of the polarisome, orchestrates tip growth in yeasts and other fungi. We identified a conserved short linear motif in the Rab GTPase-activating proteins (RabGAPs) Msb3 and Msb4, and the MAP kinase kinases Ste7 and Mkk1, which mediates their interaction with Spa2. AlphaFold predictions suggest that these initially unstructured motifs adopt an α-helical conformation upon binding to the hydrophobic cleft in the N-terminal domain of Spa2. Altering the predicted key contact residues in either Spa2 or the motif reduces complex stability. Such mutations also cause mis-localization of Msb3, Msb4 and Ste7 within the cell. Deleting the motif in Msb3 or Msb4 abolishes tip-directed growth of the yeast bud. Protein assemblies that spatially confine secretion to specific membrane regions are a common feature of eukaryotic cells. Accordingly, complexes between proteins with this motif and Spa2 were predicted in orthologs and paralogs across selected Opisthokonta, including pathogenic fungi and humans. A search for functional motifs in conformationally flexible regions of all yeast proteins identified Dse3 as a novel Spa2-binding partner.

Mitochondrial dynamics are defined by the continuous processes of fusion and fission that regulate mitochondrial shape, distribution and activity. They are also involved in cellular functions of mitochondria, such as energy production, metabolic adaptation, apoptosis and cellular stress responses. Consequently, these organelle dynamics play a crucial role in development, growth, differentiation and disease. Mitochondrial morphology is controlled by Drp1 (also known as DNM1L) and Fis1, which drive fission, whereas Opa1, Mfn1 and Mfn2 mediate fusion. The transcription, activation and degradation of these proteins are often regulated by signaling cascades that are crucial for stem cell maintenance and differentiation. In turn, mitochondrial dynamics regulate key outcomes of these pathways. We explore the interplay between mitochondrial fusion and fission proteins and such signaling pathways, including Notch, receptor tyrosine kinase, JNK, Hippo and mTOR signaling, finding that stem cell renewal and differentiation states are dependent on the regulation of signaling pathways by mitochondrial morphology and activity. Overall, this Review highlights how mitochondrial morphology and activity crucially regulate stem cell division for renewal and differentiation, examining their impact across diverse systems.

Throughout the life cycle of the unicellular parasite Trypanosoma brucei, its single flagellum remains laterally attached to the cell body by FLA and FLABP proteins, even as the parasite differentiates from the bloodstream form (BSF), found in the mammalian host, to the procyclic form (PCF), in the insect midgut. This differentiation is accompanied by changes in the dominant surface coat protein, from the variable surface glycoprotein to procyclins. There are stage-specific variants of the FLA and FLABP proteins, with FLA2 and FLA2BP found in BSFs, and FLA1 and FLA1BP in PCFs. Yet, how these proteins maintain flagellum attachment during the differentiation from BSFs to PCFs and the accompanying change in surface coat environment is unknown. Here, we used a double-induction system to test whether FLA2 and FLA2BP can maintain flagellum attachment in cells expressing procyclins. Whereas FLA2 compensated for the loss of FLA1, FLA2BP was mislocalised in PCFs and could not compensate for the loss of FLA1BP. Interestingly, when FLA2 was expressed alongside FLA2BP, FLA2BP localised to the flagellum attachment zone and flagellum attachment was maintained. Thus, we conclude that FLA2 and FLA2BP, together, will maintain flagellum attachment as the surface coat environment changes during BSF to PCF differentiation.

Actin microfilaments (F-actin) serve as tracks for myosin-driven organelle movement in plants. To understand how the F-actin network supports organelle movement, we examined the motility of peroxisomes as a common proxy for overall organelle motility in Arabidopsis thaliana. Using mutants of three villin (VLN) genes encoding major actin-bundling proteins that are actively expressed in vegetative tissues, we found that the vln4 mutation exacerbated the growth and subcellular F-actin defects in the vln2 vln3 double mutant. Compared to wild-type cells, the double and triple vln mutants exhibited progressive reduction of stable F-actin bundles and rapid remodeling of the fine filaments into a dynamic mesh. The defective F-actin organization caused significantly reduced mean speed and displacement distance of peroxisomes, although both rapid and slow movements were observed. Correlation analysis grouped complex heterogeneous peroxisome movement patterns into clusters reflecting distinct movement patterns. The vln2 vln3 vln4 triple mutant had significantly fewer peroxisomes with long-range and linear movement but produced an actin mesh network sufficient to retain basal peroxisome function. Our results provide insights into how VLN-dependent F-actin organization is coupled with the complex patterns of actomyosin-mediated organelle movement.

Chromera velia is a photosynthetic, free-living alga closely related to the apicomplexan parasites, a phylum of intracellular pathogens responsible for many devastating diseases, including malaria, cryptosporidiosis and toxoplasmosis. With molecular and cellular landmarks clearly related to but distinguishable from those found in apicomplexan parasites, Chromera provides an opportunity to investigate the evolutionary origin of the structures and processes needed for intracellular parasitism. However, tools for defining localization and functions of gene products do not exist for Chromera, which creates a major bottleneck for exploring its biology. Here we report two major advances in exploring the cell biology of this free-living relative of a large group of intracellular parasites: (1) successful cell transformation and (2) the implementation of expansion microscopy. The initial analysis enabled by these tools generated new insights into subcellular organization in different life stages of Chromera. These new developments boost the potential of Chromera as a model system for understanding the evolution of parasitism in apicomplexans.

Although many cancer cells proliferate by metabolizing extracellular proteins internalized by macropinocytosis and degraded in lysosomes, the extent to which macropinocytosis contributes to the growth of other metazoan cells remains undefined. This study analyzed macropinocytosis in proliferating murine macrophages as a mechanism for extracting amino acids from growth media. Macrophages internalized the fluid-phase probe Lucifer yellow by macropinocytosis and recycled much of it from their lysosomes by a first-order process. Inhibitors of pinocytosis inhibited cell growth. Removal of the essential amino acid leucine from growth medium reduced proliferation and allowed analysis of pinocytosis and the higher growth rates achieved by supplementation with either free leucine or bovine serum albumin (BSA) as a source of leucine. Macrophages could proliferate by macropinocytosis and digestion of BSA. In contrast, growth on free leucine exceeded the capacity of macropinocytosis to extract leucine from the medium. Dye molecules released from proteins by hydrolysis in lysosomes were recycled from cells efficiently. We propose that macropinocytosis concentrates large solutes such as proteins into lysosomes but allows amino acids and other products of lysosomal hydrolases to redistribute into macropinosomes and outside of the cell.