Journal of cell science最新文献

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.

Small secreted extracellular vesicles (EVs) mediate intercellular transport of bioactive macromolecules. How the membrane lipid phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2], which plays a crucial role in many cellular processes, impacts EV biogenesis is unclear. The primary cilium, a sensory organelle protruding from most non-dividing cells, transmits signals by shedding EVs called ectosomes. Here, we altered ciliary PI(4,5)P2 in C. elegans by manipulating the expression of the type I phosphatidylinositol 4-phosphate 5-kinase (PIP5K1) PPK-1 and deletion of the phosphoinositide 5-phosphatase (INPP5E) inpp-1, then determined the impact on release of EVs that carried cargoes tagged with fluorescent proteins. We discovered that increasing PI(4,5)P2 differentially affected ectosome shedding from distinct compartments, decreasing biogenesis of an EV subpopulation from the ciliary base, but enhancing budding from the cilium distal tip. Altering PI(4,5)P2 levels also impacted the abundance and distribution of EV cargoes in the cilium, but not the sorting of the protein cargoes into distinct subsets of ectosomes. Finally, manipulating PI(4,5)P2 did not affect cilium length, suggesting that changing PI(4,5)P2 levels can serve as a mechanism to regulate ectosome biogenesis in response to physiological stimuli without impacting cilium morphology.

Cilia and eukaryotic flagella are microtubule-based organelles that are crucial for cell motility and signaling. SAXO proteins (denoted for 'stabilizers of axonemal microtubules') are found exclusively in flagellated or ciliated organisms, but their physiological functions remain unclear. We investigated four SAXO proteins (SAXO1-SAXO4) in Chlamydomonas reinhardtii, identified via bioinformatics. All localize to cilia but differ in axonemal binding and spatial distribution. Single SAXO knockouts had no effect, whereas double mutants (saxo1/2, saxo1/3 and saxo2/3) showed shorter cilia. This phenotype intensified in the saxo1/2/3 triple mutant but not further in the quadruple mutant. Ciliary beating remained normal in saxo1/2/3 mutants, even under mechanical stress, indicating that SAXO1-SAXO3 are not essential for ciliary rigidity. Biochemical and proteomic analyses revealed no significant changes in the ciliary proteome or in tubulin acetylation, tyrosination and glutamylation within cilia. However, dikaryon assays with labeled tubulin showed that there was increased axonemal tubulin turnover in saxo1/2/3 mutant. Our findings underscore a crucial role of SAXO proteins in stabilizing axonemal microtubules by reducing tubulin turnover, thereby regulating ciliary length and assembly, and provide new insights into their function in cilia.

ARL13B is a regulatory GTPase enriched in cilia, making it a popular marker for this organelle. Arl13bhnn/hnn mice lack ARL13B expression, die during mid-gestation, and exhibit defects in ciliogenesis. The R26Arl13b-Fucci2aR biosensor mouse line directs the expression of fluorescently tagged full-length Arl13b cDNA upon Cre recombination. To determine whether constitutive, ubiquitous expression of Cerulean-tagged ARL13B (ARL13B-Cerulean) can replace endogenous gene expression, we generated Arl13bhnn/hnn animals expressing ARL13B-Cerulean. We show that Arl13bhnn/hnn;Arl13b-Cerulean mice survive to adulthood with no obvious physical or behavioral defects, indicating that the fluorescently tagged protein can functionally replace the endogenous protein during development. However, we observed that rescued males failed to sire offspring, revealing a role for ARL13B in spermatogenesis. This work shows that the R26Arl13b-Fucci2aR mouse contains an inducible allele of Arl13b capable of functioning in most tissues and biological processes.