Molecular Biology of the Cell最新文献

Electron microscopy is an important technique for the study of synaptic morphology and its relation to synaptic function. The data analysis for this task requires the segmentation of the relevant synaptic structures, such as synaptic vesicles (SV), active zones, mitochondria, presynaptic densities, synaptic ribbons, and synaptic compartments. Previous studies were predominantly based on manual segmentation, which is very time-consuming and prevented the systematic analysis of large datasets. Here, we introduce SynapseNet, a tool for the automatic segmentation and analysis of synapses in electron micrographs. It can reliably segment SVs and other synaptic structures in a wide range of electron microscopy approaches, thanks to a large annotated dataset, which we assembled, and domain adaptation functionality we developed. We demonstrated its capability for (semi-)automatic biological analysis in two applications and made it available as an easy-to-use tool to enable novel data-driven insights into synapse organization and function.

Mitochondrial membrane phospholipids impact mitochondrial structure and function by influencing the assembly and activity of membrane proteins. Although the specific roles of the three most abundant mitochondrial phospholipids, phosphatidylcholine (PC), phosphatidylethanolamine (PE), and cardiolipin (CL), have been extensively studied, the precise function of less abundant phosphatidylserine (PS) is not yet determined. Here, we used genetic and nutritional manipulation to engineer a set of yeast mutants, including a mutant completely devoid of PS, to assess its role in mitochondrial bioenergetics and lipid homeostasis. To circumvent the confounding effect of downstream PS products, PE and PC, we exogenously supplied ethanolamine that allows their biosynthesis via an alternate pathway. Using this system, we demonstrate that PS does not impact the abundance or the assembly of mitochondrial respiratory chain complexes; however, mitochondrial respiration is impaired. PS-lacking mitochondria cannot maintain mitochondrial membrane potential and exhibit leaky membranes. A mass spectrometry-based analysis of the cellular and mitochondrial lipidomes revealed an unexpected increase in odd-chain fatty acid-containing lipids in PS-lacking cells that may impact mitochondrial bioenergetics. Our study uncovers novel roles of PS in mitochondrial membrane biogenesis and bioenergetics and provides a viable eukaryotic system to unravel the cellular functions of PS.

Histone deacetylase 6 (HDAC6) helps cells manage misfolded proteins by transporting ubiquitin (UB)-associated structures toward the microtubule organizing center, where they can be sequestered and degraded by lysosomes. Here, we show that when cells are subjected to acute protein-folding stress in the endoplasmic reticulum (ER), HDAC6 depletion results in the appearance of enlarged endosomes that are highly decorated with UB and colocalize with both early and late endosome markers. The C-terminal UB-binding domain and adjacent disordered regions of HDAC6 are necessary and sufficient to rescue this endosomal phenotype in cells lacking endogenous HDAC6. HDAC6 deficiency does not appear to prevent the recruitment of endosomal sorting complexes required for transport (ESCRT), which coordinate endosome maturation. However, overexpression of HDAC6 can reverse endosome phenotypes associated with the depletion of the early ESCRT factor HRS. We speculate that HDAC6 facilitates the packaging and processing of endosomal cargo when the endomembrane system is under stress.

Hereditary spastic paraplegia type 21 (SPG21) is an inherited neurological disorder caused by biallelic mutations in the SPG21 gene, which encodes a protein named SPG21 or maspardin. Herein, we report that the SPG21 protein localizes to endolysosomes through interaction with the GTP-bound form of RAB7A. Disease-associated SPG21 variants reduce expression of SPG21 and disrupt its endolysosomal localization in both nonneuronal cells and neurons. Consistent with this localization, functional dependency analysis links SPG21 to endolysosomal and mTORC1 signaling pathways. Biochemical studies reveal that SPG21 depletion does not affect phosphorylation of canonical mTORC1 substrates such as ULK1, S6K1, 4E-BP1, but reduces phosphorylation of the noncanonical mTORC1 substrate TFEB. This enhances nuclear localization of TFEB and expression of a subset of TFEB-target genes. We conclude that SPG21 acts as a RAB7A effector that promotes noncanonical mTORC1-catalyzed phosphorylation of TFEB, thereby suppressing its nuclear localization and transcriptional activity. These findings link SPG21 dysfunction to altered endolysosomal signaling, offering new insights into SPG21 pathogenesis.

Neutrophils exert tumor-promoting roles in breast cancer and are particularly prominent in aggressive breast tumors. The proinflammatory signals TGF-β1 and TNF-α are upregulated in breast tumors and induce epithelial-to-mesenchymal transitions (EMT), a process linked to cancer cell aggressiveness. Here, we investigated the roles of TGF-β1 and TNF-α in the recruitment of neutrophils by breast cancer cells. Dual-treatment with TGF-β1 and TNF-α induces EMT signatures in premalignant M2 cells, which are part of the MCF10A breast cancer progression model. Conditioned media (CM) harvested from M2 cells treated with TGF-β1/TNF-α gives rise to amplified neutrophil chemotaxis compared with CM from vehicle-treated M2 cells. This response correlates with higher levels of the neutrophil chemokines CXCL1 and CXCL8, in a p38MAPK-dependent manner, and is attenuated by CXCL8-neutralizing antibodies. We combined gene editing, immunological, and biochemical assays to show that neutrophil recruitment and EMT are uncoupled in treated M2 cells. Finally, analysis of transcriptomic databases of cancer cell lines revealed a significant correlation between CXCL8 and TGF-β1/TNF-α-regulated or effector genes in breast cancer. These findings establish a novel role for the TGF-β1/TNF-α/p38 MAPK signaling axis in regulating neutrophil recruitment in breast cancer, independent of their profound impact on EMT.

Actin filaments play essential roles in various cellular processes, and understanding their dynamics is crucial for studying cellular behaviors and actin-related diseases. However, conventional methods for visualizing actins often perturb its functionality or lack sufficient resolution for real-time imaging. In this study, we developed a method for functional fluorescence labeling of actin isoforms using split-GFP (Green fluorescent protein) technology, specifically through insertion of a GFP11 tag into a flexible residue pair (T229/A230) of human β-actin (ACTB) or γ-actin (ACTG). This strategy (GFP11-i) was successfully applied to visualize actin dynamics in mammalian cell lines, including the effects of disease-related mutations (e.g., ACTB R196H and ACTG S155F). In addition, we demonstrated the labeling of actin filaments in Caenorhabditis elegans, further validating the cross-species applicability of this strategy. The GFP11-i methodology provides a versatile and powerful tool for investigating actin dynamics and cellular behaviors in both physiological and pathological contexts, facilitating the illustration of molecular mechanisms underlying actin-related diseases.

Transcription persists at low levels in mitotic cells and plays essential roles in mitotic fidelity and chromosomal dynamics. However, the detailed regulatory network of mitotic transcription remains largely unresolved. Here, we report the novel role of Polo-like kinase 1 (Plk1) in maintaining mitotic transcription. Using 5-ethynyl uridine (5-EU) labeling of nascent RNAs, we found that Plk1 inhibition leads to significant downregulation of nascent transcription in prometaphase cells. Chromatin-localized Plk1 activity is required for transcription regulation and mitotic fidelity. Plk1 sustains global chromosomal accessibility in mitosis, especially at promoter and transcription start site (promoter-TSS) regions, facilitating transcription factor binding and ensuring proper transcriptional activity. We identified SMC4, a common subunit of condensin I and II, as a potential Plk1 substrate. Plk1 activity is fundamental to these processes across nontransformed and transformed cell lines, underscoring its critical role in cell-cycle regulation. This study elucidates a novel regulatory mechanism of global mitotic transcription, advancing our understanding of cell-cycle control.

Escherichia coli cell shape and size are governed by the mechanochemistry of the cellular components. Inhibiting either cell-wall synthesis proteins such as FtsI leads to cell elongation and bulging, while inhibiting MreB cytoskeletal polymerization results in a loss of rod-shape. Here, we quantify cell shape dynamics of E. coli combinatorially treated with the FtsI inhibitor cephalexin and MreB inhibitor A22 and fit a shell mechanics model to the length-width dynamics to infer the range of effective mechanical properties governing cell shape. The model based on the interplay of intracellular pressure and envelope mechanics, predicts E. coli cell width grows and saturates. Bulging observed in cells treated with both MreB and FtsI inhibitors, is predicted by the model to result from a lower effective bending rigidity and higher effective surface tension compared with untreated. We validate the specificity of the predicted internal pressure of ∼0.6 MPa driving bulging, when placing treated cells in a hyperosmotic environment of comparable pressure results in reversal of cell bulging. Simulations of cell width dynamics predicting threshold values of envelope bending rigidity and effective surface tension required to maintain cell shape compared with experiment validate the effective mechanical limits of cell shape maintenance.

Guidance of cell growth or movement in response to chemical cues in the environment is critical for many cell behaviors. Budding yeast orientation of polarized growth in response to gradients of mating pheromones provides a tractable model to address how cells accurately assess small spatial differences in chemical concentrations. Pheromones bind to receptors that act through heterotrimeric G proteins to promote activation of the MAPK Fus3. Active Fus3 binds to Gα, which is thought to enhance local phosphorylation of relevant MAPK substrates to promote orientation of polarity toward high-pheromone regions. Polarity is oriented by a pathway in which Gβγ binds the scaffold protein Far1 to activate the conserved polarity regulator Cdc42, which activates the formin Bni1 to orient actin and hence growth. Gβγ, Far1, and Bni1 are all MAPK substrates whose phosphorylation could improve orientation toward high-pheromone regions. Here, we show that the Gα-MAPK interaction can enhance the efficiency of polarity-site alignment between mating partners, particularly under conditions with high Fus3 activity. Surprisingly, however, we find no evidence that phosphorylation of Gβγ, Far1, or Bni1 contribute to the benefit conferred by Gα-MAPK interaction. The precise role of this interaction remains mysterious.

Biomolecular condensates are micrometer-scale subcellular structures assembled through protein phase separation in living cells. Recent research shows that they are critical to normal biological processes and their misregulation may contribute to disease. A prominent example is the cancer-causing EML4-ALK fusion protein, which spontaneously forms biomolecular condensates that significantly enhance receptor tyrosine kinase (RTK) signaling within the condensate microenvironment. In this work, we show that a trimerization domain (TD) in EML4-ALK is necessary for condensate formation. By designing a peptide targeting the TD, we disrupted EML4-ALK self-assembly, leading to the dissolution of pre-existing EML4-ALK condensates in patient lung tumor-derived cells. Notably, this disruption significantly reduced EML4-ALK-dependent signaling and cell proliferation. Our findings demonstrate that interfering with a specific protein-protein interaction can disrupt oncogenic biomolecular condensates and attenuate their associated signaling. These results highlight the potential of targeting condensate assembly as a strategy to modulate oncogenic signaling.