Molecular Biology of the Cell最新文献

Intraflagellar transport (IFT) coordinates the transport of cargo in cilia and is essential for ciliary function. CILK1 has been identified as a key regulator of IFT. The mechanism by which it acts has, however, remained unclear. In this study, we use fluorescence imaging and single-molecule tracking in the phasmid cilia of live Caenorhabditis elegans to study the effect of the CILK1 homologue DYF-5 on the dynamics of the IFT. We show that in the absence of DYF-5, IFT components accumulate at the ciliary tip. Kinesin-II is no longer restricted to the proximal segment of the cilium but is present throughout the cilium, while its velocity is different from that of OSM-3. The frequency of IFT trains is reduced and in particular retrograde trains were rarely observed. In the absence of DYF-5, retrograde transport is vastly reduced, resulting in the accumulation of IFT components at the tip and depletion at the base. The latter results in impeded anterograde train assembly, resulting in fewer trains with irregular composition. Our results show that DYF-5 plays a key role in regulating the turnarounds of IFT trains at the ciliary tip.

The cell walls of rod-shaped Gram-positive bacteria are thick, multilayered networks that chirally twist as cells elongate. The underlying basis of twisting is not known, but probing the processes underlying this phenomenon may give insights into how cell wall material is inserted, how it evolves during cleavage, and the mechanics within the sacculus. In Bacillus subtilis, we see cell chains lacking hydrolases twist far slower than chains of wild-type cells, indicating that cell wall cleavage modulates the twisting rate. We see that when cells within chains separate, the two nascent ends rotate as they separate. Together, this suggests there is torsional stress within the cell wall that, when unreleased, perturbs overall chain morphology. Unlike Escherichia coli, we see that twisting does not arise from MreB's angle of motion, as its angle is identical in both fast-twisting wild-type cells and slow-twisting hydrolase-deficient cells. Rather, the circumferential insertion of glycans appears to establish this torsional stress, as increasing Rod complex activity by deleting ponA causes cells to twist faster than wild-type cells. Together, these experiments suggest the twisting of B. subtilis cells arises from radial glycan insertion, which somehow causes torsional stress in the wall that is later released by hydrolase activity.

Amyloid β (Aβ) inhibits hippocampal long-term potentiation (LTP; a form of synaptic plasticity thought to underly learning and memory) by inhibiting the stimulation-induced synaptic accumulation of the Ca²⁺/calmodulin (CaM)-dependent protein kinase II (CaMKII). Notably, CaMKII inhibition rescues both CaMKII movement and LTP, indicating that CaMKII mediates both LTP and the Aβ-induced LTP impairment. Somewhat counterintuitively, we found here that overexpression of GFP-CaMKII also rescued the Aβ-induced impairment of CaMKII movement. For endogenous CaMKII, we confirmed that Aβ indeed induced impairment of movement, and that previous results with live-imaging approaches were not due to Aβ-induced dissociation of the CaMKII intrabody. For exogenous GFP-CaMKII, the effect did not depend on the expression level and was thus likely caused by the N-terminal GFP label. Surprisingly, placing the GFP label instead at the C-terminus (near the association domain) still allowed CaMKII holoenzyme formation and still protected from the Aβ-induced impairment of CaMKII movement. Thus, while our method allows replacing endogenous CaMKII with similar amounts of GFP-CaMKII, our results provide a rare example for GFP-CaMKII not recapitulating the function of endogenous CaMKII.

Alzheimer's disease (AD) is characterized by the progressive spread of tau pathology throughout the brain. Inflammation has been demonstrated to be present in the disease state as well as changes in endocytic trafficking. Here we identify the Rab7 effector RILP, a protein at the intersection of inflammatory states and endocytic trafficking, as a novel player in tau propagation. We show that RILP is cleaved in AD brain and this cleavage correlates to increases in hyperphosphorylated tau. Cleavage can be induced in both BE(2) neuron-like cells as well as a microglia cell line when they are treated with the inflammatory mediators lipopolysaccharide (LPS) and ATP. This inflammatory state also enhances tau propagation between BE(2) cells, an effect that is mitigated by overexpressing a noncleavable RILP. Furthermore, microglial cells contribute to intercellular tau propagation through both the release of inflammation-associated factors and the direct uptake and secretion of tau, potentially via extracellular vesicles (EVs). In HMC3 microglial cells, RILP cleavage led to impaired tau degradation, increasing intracellular tau accumulation. Additionally, the RILP cleavage status influences EV secretion in microglia. These findings suggest that RILP cleavage alters the endocytic trafficking of tau causing increased cell-cell propagation in a cell-culture model of AD.

Specialized, maternally derived ribonucleoprotein (RNP) granules play an important role in specifying the primordial germ cells in many animal species. Typically, these germ granules are small (∼100 nm to a few microns in diameter) and numerous; in contrast, a single, extremely large granule called the oosome plays the role of germline determinant in the wasp Nasonia vitripennis. The organizational basis underlying the form and function of this unusually large membraneless RNP granule remains an open question. Here we use a combination of super-resolution and transmission electron microscopy (TEM) to investigate the composition and morphology of the oosome. We show evidence which suggests the oosome has properties of a viscous liquid or elastic solid. The most prominent feature of the oosome is a branching mesh-like network of high abundance mRNAs that pervades the entire structure. Homologues of the core germ granule proteins Vasa and Oskar do not appear to nucleate this network but rather are distributed adjacently as separate puncta. Low abundance RNAs appear to cluster in puncta that similarly do not overlap with the protein puncta. Several membrane-bound organelles, including lipid droplets and rough endoplasmic reticulum (ER)-like vesicles, are incorporated within the oosome, whereas mitochondria are nearly entirely excluded. Our findings show that the remarkably large size of the oosome is reflected in a complex subgranular organization and suggest that the oosome is a powerful model for probing interactions between membraneless and membrane-bound organelles, structural features that contribute to granule size, and the evolution of germ plasm in insects.

Sorting nexin 9 (SNX9) is a membrane-binding scaffold protein that contributes to viral uptake and inflammation and is associated with worse outcomes in several cancers. It is involved in endocytosis of epidermal growth factor receptors, β1-integrin and membrane type 1 matrix metalloprotease, and formation of mitochondrial-derived vesicles. The SNX9 Bin-Amphiphysin-Rvs (BAR)-Phox homology (PX) domains bind phosphoinositide lipids and the Src homology 3 (SH3) domain interacts with dynamin and Neural-Wiskott Aldrich syndrome protein (N-WASP) to stimulate Arp2/3 complex-mediated actin polymerization. Here we use biolayer interferometry, cell-free reconstitution, and superresolution microscopy to analyze the specificity and activities of SNX9 at membranes. We find that more SNX9 can bind liposomes containing phosphatidylinositol (4,5)-bisphosphate (PI(4,5)P₂) and phosphatidylinositol (3)-phosphate (PI(3)P) compared with phosphatidylinositol (3,4)-bisphosphate (PI(3,4)P₂), despite similar affinities. Actin assembly requires the network of both PX-BAR and SH3 interactions. Three-dimensional direct stochastic optical reconstruction microscopy on filopodia-like reconstitutions shows that SNX9 and related protein transducer of Cdc42-dependent actin assembly-1 (TOCA-1) can form both flat and ∼0.5 µm curved assemblies at actin incorporation sites. Finally, using cryo-electron tomography, we show that SNX9 builds both branched and bundled actin networks demonstrating its potential for multifunctional roles in actin remodeling.

Ceramides, especially acylceramides and protein-bound ceramides, are important for skin barrier formation. However, due to the neonatal lethality of knockout (KO) of the genes involved in the production of these ceramides, the effects of their KO in adult mice have been unclear. To investigate these effects, we created mice with tamoxifen-inducible conditional KO of the fatty acid elongase Elovl1. Following tamoxifen administration, acylceramide levels began to decrease from day 5. On day 10, impaired formation of lipid lamellae and thickening of the epidermis were observed. On day 15, protein-bound ceramide levels were substantially reduced and transepidermal water loss was increased. Changes in quantities of ceramides other than acylceramides and protein-bound ceramides and shortening of their fatty acid moieties were also observed, but time courses differed among ceramide classes. RNA sequencing revealed changes in the expression levels of genes involved in ceramide metabolism and keratinocyte proliferation and differentiation in Elovl1 conditional-KO mice. In summary, this study reveals that acylceramides and protein-bound ceramides are important for maintaining the skin barrier in adults, although they are not essential for survival. We also observed compensatory responses toward reduced skin barrier function, such as changes in gene expression, epidermal morphology, and ceramide composition.

Ras-like (Ral) GTPases play essential regulatory roles in many cellular processes, including exocytosis. Cycling between GDP- and GTP-bound states, Ral GTPases function as molecular switches and regulate effectors, specifically the multi-subunit tethering complex exocyst. Here, we show that Ral isoform RalB, but not RalA, is necessary for regulated exocytosis of Weibel-Palade bodies (WPBs), the specialized endothelial secretory granules that store hemostatic protein von Willebrand factor. Remarkably, unlike typical small GTPase-effector interactions, RalB binds exocyst in GDP-bound state. Upon endothelial cell stimulation, exocyst is uncoupled from RalB-GTP resulting in WPB tethering and exocytosis. Furthermore, we report that PKC-dependent phosphorylation of the C-terminal hypervariable region (HVR) of RalB modulates its interaction with exocyst. Exocyst preferentially interacts with phosphorylated RalB in resting endothelium. Dephosphorylation of RalB either by endothelial cell stimulation, or PKC inhibition, or expression of nonphosphorylatable mutant at a specific serine residue of RalB HVR, disengages exocyst and augments WPB exocytosis, resembling a RalB exocyst-binding site mutant. In summary, uncoupling of exocyst from RalB promotes endothelial WPB exocytosis. Our data show that RalB may be more dynamically regulated by phosphorylation and may confer distinct functionality given the high degree of homology and the shared set of effector protein between the two Ral isoforms.

Amino acid homeostasis is essential for cellular functions such as growth, metabolism, and signaling. In budding yeast Saccharomyces cerevisiae, the General Amino Acid Control (GAAC) and Target of Rapamycin Complex 1 (TORC1) pathways are utilized for intracellular amino acid sensing, while the Ssy1-Ptr3-Ssy5 (SPS) pathway is used for extracellular sensing. These pathways maintain homeostasis by responding to variations in amino acid levels to regulate amino acid biosynthesis and uptake. However, their interactions under various conditions and behavior at single-cell resolution remain insufficiently understood. We developed fluorescent transcriptional reporters to monitor amino acid biosynthesis and uptake pathways in single cells, revealing pathway engagement in response to different amino acid levels and types. Inhibition experiments demonstrated that the SPS pathway influences TORC1 and GAAC activities differently. Additionally, pathway engagement varied between liquid culture and colony environments. In colonies, some cells specialized in either amino acid synthesis or uptake. Disruption of the SPS pathway hindered this specialization and increased cell death rates in aging colonies, indicating a role for metabolic differentiation in maintaining colony viability. Collectively, this study introduces a new tool for exploring cellular amino acid homeostasis and highlights the importance of cellular differentiation in amino acid control for colony survival.

Maintaining proper tension is critical for the organization and function of the plasma membrane. To study the mechanisms by which yeast restores normal plasma membrane tension, we used a microfluidics device to expose yeast to hyperosmotic conditions, which reduced cell volume and caused a ∼20% drop in cell surface area. The resulting low tension plasma membrane exhibited large clusters of negatively-charged glycerophospholipids together with nutrient transporters, suggesting phase segregation of the membrane. We found that endocytosis was blocked by the phase segregation and thus was not involved in removing excess membrane. In contrast, rapid recovery of plasma membrane tension was dependent on 1) eisosome morphology changes that were able to absorb most of the excess surface area and 2) lipid transport from the plasma membrane to the endoplasmic reticulum (ER), where lipids were shunted into newly formed lipid droplets.