Molecular Biology of the Cell最新文献

Fluorescent proteins (FPs) are essential tools in biology. The utility of FPs depends on their brightness, photostability, efficient folding, monomeric state, and compatibility with different cellular environments. Despite the proliferation of available FPs, derivatives of the originally identified Aequorea victoria green fluorescent protein often show superior behavior as fusion tags. We recently generated msGFP2, an optimized monomeric superfolder variant of A. victoria GFP. Here, we describe two derivatives of msGFP2. The monomeric variant msYFP2 is a yellow superfolder FP with high photostability. The monomeric variant moxGFP2 lacks cysteines but retains significant folding stability, so it works well in the lumen of the secretory pathway. These new FPs are useful for common imaging applications.

Type IV P-type ATPases (P4-ATPases) are lipid flippases that generate an asymmetric membrane organization essential for cell viability. The five budding yeast P4-ATPases traffic between the Golgi complex, plasma membrane, and endosomes but how they are recycled from the endolysosomal system to the Golgi complex is poorly understood. In this study, we find that P4-ATPase endosomal recycling is primarily driven by the retromer complex and the F-box protein Rcy1. Defects in P4-ATPase recycling result in their mislocalization to the vacuole and a substantial loss of membrane asymmetry. The P4-ATPases contain multiple predicted retromer sorting signals, and the characterization of these signals in Dnf1 and Dnf2 led to the identification of a novel retromer-dependent signal, IPM[ST] that acts redundantly with predicted motifs. Together, these results emphasize the importance of endosomal recycling for the functional localization of P4-ATPases and membrane organization.

All immune cells must transit from the blood to distal sites such as the lymph nodes, bone marrow, or sites of infection. Blood borne monocytes traffic to the site of inflammation by adhering to the endothelial surface and migrating along endothelial intracellular adhesion molecule 1 (ICAM-1) by their ligand's macrophage 1 antigen (Mac-1) and lymphocyte functional antigen 1 (LFA-1) to transmigrate through the endothelium. Poor patient prognoses in chronic inflammation and tumors have been attributed to the hyper recruitment of certain types of macrophages. Therefore, targeting the binding of ICAM-1 to its respective ligands provides a novel approach to targeting the recruitment of macrophages. To that end, we determined whether the loss of Mac-1 expression could induce this upstream migration behavior by using blocking antibodies against Mac-1 to examine the effects of hydrodynamic flow on the migration of the human macrophage cell line U-937 on ICAM-1 surfaces. Blocking Mac-1 on U-937 cells led to upstream migration against the direction of shear flow on ICAM-1 surfaces. In sum, the ability of macrophages to migrate upstream when Mac-1 is blocked represents a new avenue to precisely control the differentiation, migration, and trafficking of macrophages.

Subversion of cellular membranes and membrane proliferation are used by positive-strand RNA viruses to build viral replication organelles (VROs) that support virus replication. The biogenesis of the membranous VROs requires major changes in lipid metabolism and lipid transfer in infected cells. In this work, we show that tomato bushy stunt virus (TBSV) hijacks Atg2 autophagy related protein with bulk lipid transfer activity into VROs via interaction with TBSV p33 replication protein. Deletion of Atg2 in yeast and knockdown of Atg2 in Nicotiana benthamiana resulted in decreased TBSV replication. We found that subversion of Atg2 by TBSV was important to enrich VRO membranes with phosphatidylethanolamine (PE), phosphatidylserine (PS) and PI(3)P phosphoinositide. Interestingly, inhibition of autophagy did not affect the efficient recruitment of Atg2 into VROs, and overexpression of Atg2 enhanced TBSV replication, indicating autophagy-independent subversion of Atg2 by TBSV. These findings suggest that the proviral function of Atg2 lipid transfer protein is in VRO membrane proliferation. In addition, we find that Atg2 interacting partner Atg9 with membrane lipid-scramblase activity is also coopted for tombusvirus replication. Altogether, the subversion of Atg2 bridge-type lipid transfer protein provides a new mechanism for tombusviruses to greatly expand VRO membranes to support robust viral replication.

The key bacterial cell division protein FtsZ can adopt multiple conformations, and prevailing models suggest that transitions of FtsZ subunits from the closed to open state are necessary for filament formation and stability. Using all-atom molecular dynamics simulations, we analyzed state transitions of Staphylococcus aureus FtsZ as a monomer, dimer, and hexamer. We found that monomers can adopt intermediate states but preferentially adopt a closed state that is robust to forced reopening. Dimer subunits transitioned between open and closed states, and dimers with both subunits in the closed state remained highly stable, suggesting that open-state conformations are not necessary for filament formation. Mg²⁺ strongly stabilized the conformation of GTP-bound subunits and the dimer filament interface. Our hexamer simulations indicate that the plus end subunit preferentially closes and that other subunits can transition between states without affecting inter-subunit stability. We found that rather than being correlated with subunit opening, inter-subunit stability was strongly correlated with catalytic site interactions. By leveraging deep-learning models, we identified key intrasubunit interactions governing state transitions. Our findings suggest a greater range of possible monomer and filament states than previously considered and offer new insights into the nuanced interplay between subunit states and the critical role of nucleotide hydrolysis and Mg²⁺ in FtsZ filament dynamics.

The regulation of maternal mRNAs is essential for proper oogenesis, the production of viable gametes, and to avoid birth defects and infertility. Many oogenic RNA-binding proteins have been identified with roles in mRNA metabolism, some of which localize to dynamic ribonucleoprotein granules and others that appear dispersed. Here, we use a combination of in vitro condensation assays and the in vivo Caenorhabditis elegans oogenesis model to characterize the properties of the conserved KH-domain MEX-3 protein and to identify novel regulators of MEX-3 and three other translational regulators. We demonstrate that MEX-3 undergoes phase separation and appears to have intrinsic gel-like properties in vitro. We also identify novel roles for the chaperonin-containing tailless complex polypeptide 1 (CCT) chaperonin and actin in preventing ectopic RNA-binding protein condensates in maturing oocytes that appear to be independent of MEX-3 folding. The CCT chaperonin and actin also oppose the expansion of endoplasmic reticulum sheets that may promote ectopic condensation of RNA-binding proteins. These novel regulators of condensation are also required for the translational repression of maternal mRNA which is essential for oocyte quality and fertility. The identification of this regulatory network may also have implications for understanding the role of hMex3 phase transitions in cancer.

Genetic, colocalization, and biochemical studies suggest that the ankyrin repeat-containing proteins Inversin (INVS) and ANKS6 function with the NEK8 kinase to control tissue patterning and maintain organ physiology. It is unknown whether these three proteins assemble into a static "Inversin complex" or one that adopts multiple bioactive forms. Through the characterization of hyperactive alleles in C. elegans, we discovered that the Inversin complex is activated by dimerization. Genome engineering of an RFP tag onto the nematode homologues of INVS (MLT-4) and NEK8 (NEKL-2) induced a gain-of-function, cyst-like phenotype that was suppressed by monomerization of the fluorescent tag. Stimulated dimerization of MLT-4 or NEKL-2 using optogenetics was sufficient to recapitulate the phenotype of a constitutively active Inversin complex. Further, dimerization of NEKL-2 bypassed a lethal MLT-4 mutant, demonstrating that the dimeric form is required for function. We propose that dynamic switching between at least two functionally distinct states - an active dimer and an inactive monomer - gates the output of the Inversin complex.

Morphological complexity and plasticity are hallmarks of polyextremotolerant fungi. Septins are conserved cytoskeletal proteins and key contributors to cell polarity and morphogenesis. They sense membrane curvature, coordinate cell division, and influence diffusion at the plasma membrane. Four septin homologues are conserved from yeasts to humans, the systems in which septins have been most studied. But there is also a fifth family of opisthokont septins that remain biochemically mysterious. Members of this family, Group 5 septins, appear in the genomes of filamentous fungi, but are understudied due to their absence from ascomycete yeasts. Knufia petricola is an emerging model polyextremotolerant black fungus that can also serve as a model system for Group 5 septins. We have recombinantly expressed and biochemically characterized KpAspE, a Group 5 septin from K. petricola. This septin--by itself in vitro--recapitulates many functions of canonical septin hetero-octamers. KpAspE is an active GTPase that forms diverse homo-oligomers, binds shallow membrane curvatures, and interacts with the terminal subunit of canonical septin hetero-octamers. These findings raise the possibility that Group 5 septins govern the higher-order structures formed by canonical septins, which in K. petricola cells form extended filaments, and provide insight into how septin hetero-oligomers evolved from ancient homomers.

Adaptation to environmental stress requires coordination between stress-defense programs and cell cycle progression. The immediate response to many stressors has been well characterized, but how cells survive in challenging environments long term is unknown. Here, we investigate the role of the stress-activated phosphatase calcineurin (CN) in adaptation to chronic CaCl₂ stress in Saccharomyces cerevisiae. We find that prolonged exposure to CaCl₂ impairs mitochondrial function and demonstrate that cells respond to this stressor using two CN-dependent mechanisms-one that requires the downstream transcription factor Crz1 and another that is Crz1 independent. Our data indicate that CN maintains cellular fitness by promoting cell cycle progression and preventing CaCl₂-induced cell death. When Crz1 is present, transient CN activation suppresses cell death and promotes adaptation despite high levels of mitochondrial loss. However, in the absence of Crz1, prolonged activation of CN prevents mitochondrial loss and further cell death by upregulating glutathione biosynthesis genes thereby mitigating damage from reactive oxygen species. These findings illustrate how cells maintain long-term fitness during chronic stress and suggest that CN promotes adaptation in challenging environments by multiple mechanisms.

All cells must detect, interpret, and adapt to multiple and concurrent stimuli. While signaling pathways are highly specialized, different pathways often share components or have components with overlapping functions. In the yeast Saccharomyces cerevisiae, the high osmolarity glycerol (HOG) pathway has two seemingly redundant branches, mediated by Sln1 and Sho1. Both branches are activated by osmotic pressure, leading to phosphorylation of the MAPKs Hog1 and Kss1. The mating pathway is activated by pheromone, leading to phosphorylation of the MAPKs Fus3 and Kss1. Given that Kss1 is shared by the two pathways, we investigated its role in signal coordination. We activated both pathways with a combination of salt and pheromone, in cells lacking the shared MAPK and in cells lacking either of the redundant branches of the HOG pathway. By systematically evaluating MAPK activation, translocation, and transcription programs, we determined that Sho1 mediates cross talk between the HOG and mating pathways and does so through Kss1. Further, we show that Kss1 initiates a transcriptional program that is distinct from that induced by Hog1 and Fus3. Our findings reveal how redundant and shared components coordinate concurrent signals and thereby adapt to sudden environmental changes.