Molecular Biology of the Cell最新文献

Transient transfection is widely used for protein expression in heterologous systems, yet uncontrolled overexpression frequently introduces artifacts that confound functional analyses. Although stable cell lines can mitigate these issues, generating lines for multiple constructs or variants is often impractical. Common alternatives, such as DNA titration, altered transfection conditions, or promoter swapping, provide only coarse and inconsistent control of protein abundance. Here, we establish a panel of 10 human 5' untranslated regions (5'UTRs) as a modular strategy to tune protein expression during transient transfection. Across three soluble proteins and three membrane proteins, these 5'UTRs produce a reproducible dynamic range of expression, including fine-grained control of eYFP and the large sensory ion channel TRPA1. Notably, one 5'UTR consistently suppresses expression across all proteins tested and alleviates overexpression-associated artifacts, improving functional analysis of a hyperactive channel variant, substantially reducing background in proximity biotinylation assays, and enhancing the specificity of a stress granule marker. In contrast, most 5'UTRs enhance expression of the TRPV1 and TRPM8 sensory receptors, improving protein yield in heterologous systems. Together, this work identifies 5'UTRs as a compact, versatile, and broadly applicable tool to fine-tune protein abundance, enabling more physiologically relevant and assay-optimized expression in transient transfection experiments.

Accurate mitosis is essential for genome stability. Although dynamin is best known for its role in vesicular trafficking, its functions during mitosis remain unclear. Here, we identify a mitotic role for the Caenorhabditis elegans dynamin homolog DYN-1 in regulating spindle assembly and the localization of the key mitotic regulators SPD-2 and PLK-1. Depletion of DYN-1 results in enlarged metaphase spindle poles and increased centrosomal SPD-2 and PLK-1 levels. These effects likely depend on DYN-1-mediated vesicular trafficking, as shown by assays with the dyn-1(ky51) allele. Additionally, a subset of DYN-1-depleted embryos exhibit altered PLK-1 localization at the midbody during late mitosis, correlating with midbody assembly defects. Together, these findings establish DYN-1 as a previously unrecognized regulator of spindle pole organization and early midbody assembly.

Cortical excitability, a phenomenon in which the cell cortex is dynamically patterned with waves of F-actin assembly, has been described in a variety of model systems, including embryos of mammals, flies, frogs, and echinoderms, as well as a variety of cultured cells. While the cortical F-actin network is closely linked with the plasma membrane, it is not known if membrane composition or fluidity regulates dynamic cytokinetic patterning. Phospholipids partition within the plasma membrane during cytokinesis and phosphoinositides play a key regulatory role in other excitable systems, suggesting a role for membrane-dependent regulation of cytokinetic patterning. Here, we use an artificial cell cortex comprised of Xenopus laevis egg extract and supported lipid bilayers (SLBs) to show that membrane composition regulates self-organized cortical patterning. We find that manipulating the levels of candidate lipids, including phosphatidylinositol 4,5-bisphosphate, phosphatidylethanolamine, sphingomyelin, and cholesterol, changes the dynamics of traveling waves and standing oscillations of active Rho and F-actin, as well as the kinetics of Rho activation and F-actin assembly on SLBs. Our findings demonstrate that membrane composition regulates the assembly of cortical F-actin, as well as emergent active Rho and F-actin patterning.

The use of fluorescent proteins has become ubiquitous throughout the life sciences as a key method for understanding molecular, cellular, and tissue-scale processes. Genetically encodable fluorophores have enabled stable genetic lines to be generated in a large array of organisms. There are now suites of fluorophores available, particularly in the green and red spectra. Yet, which fluorophore works best in vivo can depend on a range of factors, both extrinsic (e.g., pH and temperature) and intrinsic (e.g., photobleaching and brightness). While fluorophores have been well characterised in cell culture, such measures within in vivo systems are more limited. Here, we present a quantitative screen of nine green and eight red fluorophore lines in Drosophila, with the fluorescent protein expressed from the same genomic location and imaged under identical conditions. We analyse the expression of the fluorophores in both early and late Drosophila embryos. We provide a quantitative analysis of the bleaching and folding rates. We find amongst the green fluorophores that the suitable choice-e.g., mEGFP, mNeonGreen, StayGold-E138D-depends on timing and imaging requirements. For the red fluorophores, mScarlet-I performed consistently well, though no particular fluorophore stood out as ideal under all conditions. These results provide a powerful database for selecting optimal fluorophores for imaging in the Drosophila embryo in green and red channels.

Many invertebrates have obliquely striated muscles, in which neighboring thin and thick filaments are staggered and aligned obliquely. This type of muscle allows force production over a wide range of lengths and is beneficial for soft-bodied animals. Unlike vertebrate cross-striated muscles, most of the obliquely striated muscles lack distinct Z-lines and, instead, have dense bodies. Because the dense bodies are located in the middle of the I-bands and contain α-actinin, the dogma is that dense bodies are equivalent to the Z-lines anchoring the actin barbed ends. However, we show that the barbed ends of sarcomeric actin filaments in the nematode Caenorhabditis elegans body wall muscle are aligned linearly without converging at the dense bodies. Colocalization of F-actin and ATN-1/α-actinin was negatively correlated. CAP-1, an α-subunit of capping protein/CapZ, was linearly aligned without concentration at the dense bodies independently of ATN-1. Depletion of the capping protein subunit, CAP-1 or CAP-2, caused embryonic or larval lethality with severe actin disorganization in muscle, indicating that barbed-end regulation by capping protein is essential for sarcomere assembly. These results contradict the current view of the sarcomere organization in C. elegans muscle and suggest a new model of a linear Z-line-like arrangement of actin barbed ends.

Aging is a complex biological process that heightens susceptibility to age-related diseases, often driven by declining mitochondrial function. Mitophagy, the selective removal of damaged mitochondria, is a key quality-control mechanism essential for maintaining cellular health, and its decline has been closely linked to aging. However, the specific role of mitophagy in cellular senescence, a hallmark of aging, remains insufficiently understood, largely due to the lack of methods to manipulate mitophagy. In this study, we used UMI-77, a new potent mitophagy activator, to evaluate its effects on senescence in mouse mesenchymal stem cells (MSC). Our results show that UMI-77 preserves mitochondrial integrity and effectively delays cellular senescence through mitophagy. Mechanistically, UMI-77 markedly suppressed the senescence-associated secretory phenotype (SASP). Together, our findings reveal a new antiaging therapeutic application for UMI-77 by targeting senescence-associated chronic inflammation through mitophagy induction and SASP reduction.

The function of p12, the smallest subunit of human DNA polymerase delta (Polδ), in DNA replication and DNA damage response (DDR) is poorly understood. Due to the identification of two protein encoding p12 isoforms, the respective Polδ variants may function differently; therefore, here we explored their cellular roles in genome maintenance. p12 isoform-1 level remains unchanged along with the catalytic subunit of Polδ throughout the cell cycle stages and upon DNA damage. Cells with a low level of Polδ or p12-depleted Polδ exhibited a slower cell cycle progression, reduced proliferation rate, and higher susceptibility to genotoxic agents and PARP1 inhibitors. Loss of p12 activated checkpoints and altered DDR proteins expression. DNA fiber assays ascertained higher accumulation of stalled and collapsed replication forks in p12 depleted cells. Similar to the dimerization and PCNA interaction defective p12 isoform-1 mutants, p12 isoform-2, which intrinsically lacks the PIP motif, failed to rescue the p12 genomic loss. p12 isoform-2 overexpression enhanced the cytotoxicity of DNA-damaging agents in cells that express a low amount of isoform-1. Thus, p12 isoform-2 seems to possess a negative dominance phenotype in wild type cells. Altogether, our results revealed distinct roles of p12 isoforms in regulating Polδ's function in genome stability.

Orsay virus (OV) is a positive-sense, single-stranded RNA (+ssRNA) virus that naturally infects Caenorhabditis elegans intestines. Like other +ssRNA viruses, the OV-encoded RNA-dependent RNA polymerase (oRdRP) synthesizes complementary antigenome for use as template for amplifying viral genome, but OV replication intermediates are underexplored. Using PCR, we observed viral genome in vast excess of antigenome, as for other +ssRNA viruses. Unlike interferon-based antiviral defense, C. elegans utilizes RNA interference (RNAi) for antiviral defense, producing sense and antisense small interfering RNAs (siRNAs) that cannot be distinguished from genome and antigenome with conventional hybridization protocols. Fluorescence-based imaging in C. elegans intestines using probes to antigenomic sequences revealed cytoplasmic as well as perinuclear localization patterns. The latter depended on factors required for generation of primary, but not secondary, siRNAs, connecting the antigenomic hybridization pattern to RNAi. We also observed cytoplasmic double-stranded RNA (dsRNA) associated with oRdRP, suggesting viral replication hubs, as well as infection-induced nuclear dsRNA, likely from endogenous dsRNA. Finally, using antibodies to oRdRP, we observed spherical structures of ∼1µm in diameter with oRdRP at their surface, which decrease in animals lacking RDE-1. Our study defines features of OV replication intermediates, setting the stage for understanding their connection to host antiviral pathways.

Autophagy is an intracellular degradation process that maintains homeostasis, responds to stress, and plays key roles in preventing aging and disease. Autophagosome biogenesis, vesicle rocketing, and autolysosome tubulation are controlled by multiple actin cytoskeletal factors, but the impact of actin assembly on completion of the autophagic degradation pathway is not well understood. Here we studied autophagosomes and lysosomes in mouse fibroblasts harboring an inducible knockout (iKO) of the Arp2/3 complex, an essential actin nucleator. Arp2/3 complex ablation resulted in increased basal levels of autophagy receptors and lipidated membrane proteins from the LC3 and GABARAP families. Such phenotypes were accompanied by the steady-state presence of abnormally high numbers of autolysosomes and an inability of the Arp2/3 complex-deficient cells to complete autolysosome turnover due to lysosomal damage. When normal cells were treated with a lysosomal membrane-disrupting agent, the Arp2/3-activating protein WHAMM was recruited to lysosomes, and Arp2/3 complex activity was required for restoring intact lysosomal structure. Deletion of WHAMM in mouse or human fibroblasts decreased Arp2/3 localization to lysosomes and increased lysosomal damage. These results reveal the importance of the Arp2/3 complex and WHAMM for autophagic degradation and uncover a new role for the actin nucleation machinery in maintaining lysosomal integrity.

Cells maintain an appropriate size to function, yet the mechanisms that enable size adaptation to environmental stress remain poorly understood. Fission yeast cells enter mitosis and divide at a threshold size when cyclin-dependent kinase (Cdk1) is activated through size- and time-dependent scaling of its regulators: Cdr2 kinase with cell surface area (SA), Cdc25 phosphatase with cell volume, and mitotic cyclin Cdc13 with time. This integrated size control network is characterized in nutrient-rich conditions, but under stress, it remains unclear which size parameters cells monitor, and which size- or time-sensing pathways mediate cell size changes. Using high-throughput image analysis, we quantified the geometry of dividing cells under osmotic, oxidative, and low glucose conditions. Wild-type cells increased their SA-to-volume (SA:Vol) ratio in low glucose but decreased it under osmotic or oxidative stress, revealing distinct stress-specific geometric responses. Genetic perturbations of size- and time-sensing pathways revealed that Cdc25 is required for volume-based expansion in oxidative and osmotic stress, Cdr2 promotes SA-based expansion in low glucose, and Cdc13 contributes to geometry changes under low glucose and osmotic stress. Although disrupting individual pathways altered normal geometric responses, cells remained viable, suggesting that a modular size control system enables flexible geometric responses to changing environments.