Journal of cell science最新文献

Krüppel-like factor 1 [KLF1; also known as erythroid Krüppel-like factor (EKLF)] is a C2H2 zinc finger transcription factor that plays a crucial role in all aspects of erythropoiesis. Mutations in KLF1 lead to diverse phenotypes ranging from mild to severe anemias. Individuals with a heterozygous E325K mutation [congenital dyserythropoietic anemia (CDA) type IV] exhibit impaired erythroid terminal differentiation and increased presence of binucleate erythroblasts. We have previously shown that KLF1 is necessary for cell cycle exit and enucleation in mouse primary cells. In the present study, we discovered that genes involved in cell motility, cell division and mitotic pathways are all directly regulated by KLF1. Klf1-/- cells exhibit increased numbers of binucleated erythroblasts and DNA bridges, and differentiating Klf1-/- erythroblasts display an increased percentage of cytokinesis failure events and defective microtubule bundling. Klf1-/- erythroblasts produce frequent aberrant F-actin-rich membrane protrusions and anucleate cell fragments. Human CDA type IV cells exhibit similar patterns of dysregulation of cytokinesis and cell motility genes. Collectively, we show that KLF1 is necessary for maintaining the integrity of erythroid cell divisions by direct regulation of genes involved in cytokinesis and motility pathways during terminal erythroid differentiation.

Hormones typically regulate physiology by modulating transcriptional programmes. However, post-transcriptional mechanisms offer an additional layer of control, enabling rapid and context-specific regulation of gene expression. Among these mechanisms, cytoplasmic ribonucleoprotein granules (RNPGs) - a type of membraneless condensate that includes stress granules and processing bodies - have emerged as dynamic regulators of RNA fate. These granules could serve as integrative hubs that modulate mRNA translation, stability and storage in response to endocrine signals, thereby fine-tuning hormone-driven cellular responses. This Hypothesis article proposes that hormonal cues can influence RNPG assembly, composition and physical state through transcriptional regulation of granule components or via rapid, non-genomic mechanisms, including kinase cascades or ligand-induced conformational changes in granule proteins. In turn, RNPGs can regulate hormone-driven cellular responses by selectively sequestering, releasing or degrading specific mRNAs. Furthermore, these granules can regulate hormonal pathways by controlling the availability of hormone-related transcripts and signalling components, establishing a bidirectional regulatory network. This dynamic interaction, illustrated by examples from plants, invertebrates and mammals, is hypothesised to add complexity and versatility to endocrine regulation, enabling rapid and adaptive responses to physiological demands.

We present evidence that the association of the epithelial (E)-cadherin (CHD1) extracellular domain and epidermal growth factor receptor (EGFR, ErbB1) is obligatory for cadherin force transduction signaling. E-cadherin and EGFR associate at cell surfaces, independent of their cytoplasmic domains, and tension on E-cadherin activates EGFR signaling. Using engineered E-cadherin mutants that disrupt co-immunoprecipitation with EGFR, but not adhesion, we show that the hetero-receptor complex is required to mechanically activate signaling and downstream cytoskeletal remodeling at cadherin adhesions. The mutants localized the essential region on E-cadherin to domain 4 of the extracellular region (EC4). The ectodomain is also required for hetero-receptor colocalization at intercellular junctions. Although the E-cadherin mutants disrupt EGFR signaling, integrin pre-activation together with tension rescues cytoskeletal reinforcement at cadherin adhesions, confirming the role of integrins in intercellular force transduction. Furthermore, although E-cadherin suppresses EGFR-mediated proliferation, in response to extracellular matrix stiffening, the force-sensitive hetero-receptor complex regulates growth factor-dependent epithelial proliferation. These findings support the hypothesis that E-cadherin complexes with EGFR are mechano-switches at cell-cell contacts that directly couple intercellular force fluctuations to mitogen-dependent signaling.

The nucleus must maintain shape and integrity to protect the function of the genome. Nuclear blebs are deformations identified by decreased DNA density that commonly lead to rupture. Lamin B levels often vary drastically between blebs. We tracked rupture via time-lapse imaging of nuclear localization sequence (NLS)-GFP immediately followed by immunofluorescence imaging of lamins and known rupture markers. We find that lamin B1 loss consistently marks ruptured nuclear blebs better than lamin A/C, emerin and cGAS. Visualizing post-rupture lamin B1 loss and emerin enrichment reveals that cell lines display widely different propensities for nuclear bleb rupture. To determine how rupture affects DNA damage, we time-lapse-imaged ruptured and unruptured blebs, then conducted immunofluorescence on the same cells for DNA damage markers γH2AX and 53BP1. We find that DNA damage is increased in blebbed nuclei independently of rupture. This was verified in blebbed LNCaP nuclei, which do not rupture and maintain lamin B1, but still show increased DNA damage. Thus, we confirm that lamin B is the most consistent marker of nuclear rupture, and that blebbed nuclei have increased DNA damage regardless of rupture.

Cancer cells adapt to external biophysical cues, but how cytoskeletal remodeling facilitates this mechano-adaptation is largely unexplored. Here, we demonstrate that intrinsic non-muscle myosin II (NMII) activity and self-organization in cancer cells regulate cellular elastic properties when cells are exposed to fluid shear stress (FSS). In association with the reorganized actin filament network, NMII bipolar filaments can assemble into aligned stacks, which allow cellular stretching upon exposure to FSS. Inhibition of NMII by treatment with small interfering RNA, (-)blebbistatin or Y27632 impairs the stack formation and perturbs cellular elasticity. Moreover, NMII-mediated elasticity regulates cyto-nuclear coupling through its association with the LINC complex protein nesprin2 and regulates nuclear import of the mechanoresponsive proteins YAP1 and TAZ (also known as WWTR1), which induce differential expression of genes thus decreasing growth and migration in FSS-exposed cells. These findings reveal that the cellular elasticity mediated by NMII dynamics provides mechano-adaptation against a mechanical stress, like FSS.

Uncoordinated-45 (UNC45) is a conserved protein required for myosin accumulation during muscle development. Invertebrates have one unc-45 gene whereas vertebrates have two paralogs, UNC45A and UNC45B, which exhibit different expression patterns. We used the Drosophila model to investigate the ability of the vertebrate proteins to function in an invertebrate system, as well as the potential evolutionary redundancy of its human paralogs. Transgenic expression of either human UNC45 paralog early in indirect flight muscle development resulted in impaired flight, disordered muscle organization and unique sub-sarcomere localizations. We then generated chimeric proteins that replaced each of three Drosophila Unc-45 domains with their human cognates. We found that a chimera containing the myosin-binding UCS domain of human UNC45A impaired muscle function, whereas none of the UNC45B domain chimeras significantly impacted flight ability. Overall, our study shows that there is significant evolutionary divergence between vertebrate and invertebrate paralogs and that the human proteins differentially disrupt Drosophila myofibril assembly and function, suggesting that they are functionally unique.

Mitochondrial dynamics relies on the function of dynamin family GTPase proteins including mitofusin 1 (MFN1), mitofusin 2 (MFN2) and dynamin-related protein 1 (DRP1; also known as DNM1L). The mitochondrial phosphatase phosphoglycerate mutase 5 (PGAM5) protein can regulate the phosphorylation levels and the function of both MFN2 and DRP1; however, the precise regulation of PGAM5 activity is unknown. Here, we show that PGAM5 oligomerization and localization controls its function. Under depolarization and/or metabolic stress PGAM5 changes its association and, instead of forming dodecamers, forms dimers. These PGAM5 oligomers have differential affinity towards MFN2 and DRP1. Simultaneously, PGAM5 is cleaved by the inner mitochondrial membrane-resident proteases PARL and OMA1 and a fraction of the cleaved PGAM5 translocates to the cytosol. These two events play an important role in regulating mitochondrial dynamics under depolarization and/or metabolic stress. Taken together, our results identify PGAM5 oligomerization and cleavage-induced relocalization as crucial regulators of its function.

We present the first three-dimensional time-resolved imaging of the Chlamydomonas reinhardtii flagellar waveform. This freshwater alga is a model system for eukaryotic flagella that allow cells to move and pump fluid. During the power stroke, the flagella show rotational symmetry about the centre line of the cell, but during the recovery stroke they display mirror symmetry about the same axis. Furthermore, and in contrast to the usual assumptions about beat planarity, we show a subtle rotational motion of the flagella at the initiation of the power stroke, which is mechanically rectified into a quasi-planar mode. We apply resistive force theory to infer the swimming speed and rotational speed of the cells, when a force-free configuration is approximated using a cell on a micropipette, showing good agreement with experimental results on freely swimming cells.

Functional adipose tissue is essential for maintaining systemic metabolic homeostasis. Dysfunctional adipose tissue, characterized by increased fibrosis, hypoxia and chronic inflammation, is often associated with obesity and promotes the onset of metabolic disease, such as type 2 diabetes. During nutrient excess, adipose tissue function can be preserved by the generation of new adipocytes from adipocyte stem cells, illustrating the importance of identifying the physiological regulators of adipogenesis. Here, we discover a cilia-localized signaling pathway through which the pro-inflammatory lipid metabolite prostaglandin E2 (PGE2) suppresses adipogenesis. We demonstrate that PGE2 specifically signals through the E-type prostaglandin receptor 4 (EP4) localized to the primary cilium of adipocyte stem cells. Activation of ciliary EP4 initiates a cAMP-independent signaling cascade that activates Rho-associated protein kinase 2 (ROCK2), resulting in the retention of actin stress fibers that prevent adipogenesis. These findings uncover a compartmentalized regulatory mechanism of adipogenesis by which primary cilia alter whole-cell physiology, cell fate, and ultimately adipose tissue expansion in response to an inflammatory hormone, offering insight into how chronic inflammation may contribute to adipose tissue dysfunction and metabolic disease progression.

Pathogenic variants in KATNIP (encoding katanin-interacting protein) are linked to Joubert syndrome, a prototypical ciliopathy. KATNIP is a scaffold protein that binds and potentiates ciliogenesis-associated kinase 1 (CILK1) activation and function to control cilia length and frequency. We previously showed that of the three predicted 'domains of unknown functions' (DUFs) in KATNIP, the DUF2 domain alone supports binding to CILK1 without activating CILK1. Here, we report three human disease variants of KATNIP with different lengths that exhibit loss of function. The longest variant of KATNIP M1474C, which is truncated near the C-terminus, binds to CILK1 but does not support the activating TDY phosphorylation in CILK1, the phosphorylation of CILK1 substrates, or the restriction of cilia length and ciliation rate. Deletion analysis of KATNIP further revealed that residues 1524-1573 encompassing predicted β-sheets and an α-helix are essential for CILK1 activation and function. The results support a model where KATNIP uses separate domains to bind and to enhance activation of CILK1, enabling CILK1 function in control of cilia formation and elongation.