Molecular Biology of the Cell最新文献

The small hypercondensed sperm nucleus undergoes a dramatic transformation into a large, round pronucleus with relaxed chromatin during the brief cleavage period in metazoan embryos, enabling the activation of chromatin functions necessary for subsequent development. However, it remains unclear whether the egg cytoplasm-specific physicochemical properties play a role in pronucleus assembly. Here, we evaluated the impact of abundant RNAs in eggs on pronucleus assembly in the Xenopus laevis cell-free reconstitution system. We found that the introduction of RNAs at an appropriate concentration led to a rapid nuclear growth, more dispersed chromatin distribution, and dissociation of sperm-specific nuclear proteins from the chromatin. These chromatin remodeling properties, which were reproducible through the introduction of negatively charged compounds, facilitated the incorporation of somatic histones into chromatin in the reconstituted nuclei. Based on these findings, we propose that the remodeled chromatin by negatively charged cytoplasmic RNAs accelerates rapid decondensation of negatively charged chromatin and pronucleus assembly during the brief cleavage period following fertilization.

The ubiquitous and highly conserved programmed cell death pathways that are essential for tissue development and homeostasis are accompanied by distinct morphological alterations. Apoptotic cells undergo fragmentation that is concomitant with the exposure of phosphatidylserine (PS) on the membrane surface. Large fragments, called apoptotic bodies, as well as much smaller and more numerous vesicles, are released. While the molecular mechanisms underlying apoptotic body formation have been explored, much less is known about vesicle biogenesis. We used an inducible, active form of TMEM16F to determine the role of lipid scrambling in vesiculation, separately from other apoptotic signaling events. Plasmalemmal lipid scrambling sufficed to release apoptotic-like vesicles without causing changes in cytosolic calcium or the submembrane cytoskeleton. The scrambled bilayer showed pronounced segregation of exofacial lipids and redistribution of detectable cholesterol to the inner leaflet. The clustering of raft-associated components with bulky headgroups-typified by glycophosphatidylinositol-linked proteins-formed domains of outward (convex) curvature, while regions of accumulation of phosphatidylethanolamine (PE) generated inward (concave) curvature that facilitated the scission of vesicles. Thus, scrambling of plasma membrane lipids suffices to induce regions of acute membrane curvature and facilitates detachment of vesicles analogous to those released from the surface of apoptotic cells.

Rab GTPases are key regulators of endosomal trafficking in eukaryotes. In mammalian cells, Rab4 and Rab7 were shown to localize to distinct compartments, with Rab4 on early endosomes for fast recycling and Rab7 on late endosomes for degradation. Here, we show that in Drosophila, endogenous Rab4 and Rab7 extensively colocalize across tissues and developmental stages. Recruited to the same compartments through mechanisms that do not require the activity of the other, they have opposing effects on endolysosomal size: Rab4 overexpression or Rab7 impairment leads to enlarged endolysosomes, whereas Rab4 loss or constitutively active Rab7 reduces their sizes. Rab4 deficiency suppresses the swelling induced by Rab7 impairment, and conversely, Rab7 activation mitigates the swelling induced by Rab4 overexpression. Genetically, Rab4 loss selectively compromises the viability of Rab7-deficient flies but not Rab5 or Rab11 mutants, supporting a functional overlap between Rab4 and Rab7. Moreover, the levels of endogenous βPS-Integrin, a cargo recycled by Rab4 and degraded via Rab7, are elevated in rab4 mutants and reduced with Rab4 overexpression. Lastly, Rab4 and Rab7 show notable colocalization in mammalian cells and mouse brains, and live imaging reveals dynamic β1-integrin trafficking between Rab4- and Rab7-positive endosomes. Together, these data support that in addition to recycling, Rab4 plays a role in degradation by directing its cargos such as β1-integrin into Rab7-mediated late endolysosomal pathway.

The mechanical properties of cells are governed by the cytoskeleton, a dynamic network of actin filaments, intermediate filaments, and microtubules. Understanding the individual and collective mechanical contributions of these three different cytoskeletal elements is essential to elucidate how cells maintain mechanical integrity during deformation. Here, we use a custom single-cell rheometer to identify the distinct contributions of actin and vimentin to the viscoelastic and nonlinear elastic response of cells to uniaxial compression. We used mouse embryonic fibroblasts (MEF) isolated from wild-type (WT) and vimentin knockout (vim^-/-) mice in combination with chemical treatments to manipulate actin polymerization and contractility. We show through small amplitude oscillatory measurements and strain ramp tests that vimentin, often overlooked in cellular mechanics, plays a role comparable with actin in maintaining cell stiffness and resisting large compressive forces. However, actin appears to be more important than vimentin in determining cellular energy dissipation. Finally, we show by comparing WT and enucleated cells that compression stiffening originates from the actin and vimentin cytoskeleton, while the nucleus appears to play little role in this. Our findings provide insight into how cytoskeletal networks collectively determine the mechanical properties of cells, providing a basis to understand the role of the cytoskeleton in the ability of cells to resist external as well as internal forces.

As with many cell types, macrophages are sometimes filled with micron-sized lipid droplets (LD's), but effects on phagocytosis of other cells, particulates, and microbes remain unclear. Here, we show that LDs restructure the cytoskeleton but remain round, consistent with a high interfacial tension; functionally, LD's impair actomyosin-driven uptake, which proves independent of target size. Engulfment of targets starts at the apical surface, but LD's displace apical actomyosin to the basal cortex. Partial rescue occurs tissue-relevant compressive stresses which activate actomyosin. Macrophages that are densely filled with LD's or pre-engulfed rigid beads likewise activate actomyosin, which again rescues phagocytosis relative to sparsely loaded cells. As further evidence of LD rigidity, both LD's and rigid beads impede macrophage migration through small pores, and LD's pressed into a nucleus cause rapid focal rupture independent of actin. LD rigidity thus disrupts cytoskeleton organization and nucleus integrity, suppressing motility processes unless actomyosin is activated by cell compression or stretching.

Intracellular microtubule-based transport depends on an essential plus-ended molecular motor, kinesin-1. The N-terminal ATP-dependent head driving motility and a C-terminal cargo interacting tail, both bind microtubules. Previously, the interplay of both domains were hypothesized to play a role in collective microtubule sliding patterns, but the mechanisms remained unclear. Here, we show that full-length Drosophila kinesin-1 results in spontaneous spatiotemporal patterns in gliding assays including bending, looping, and oscillations as well as stop-and-go motion of microtubules. We demonstrate that presence of the motor domain alone cannot produce these patterns. The tail itself can bind microtubules passively, acting as a static "anchor." An equimolar mixture of these two domain constructs reproduces the spatiotemporal patterns both in terms of increased bending with length and velocity distributions. Based on a simple "coin-toss" model, where either the head or tail bind microtubules with equal probability, we test the effect of increasing proportions of motor and tail and find antagonistic interactions drive velocity distributions with coexistence of bimodal distributions when the two tendencies are balanced. This mechanical effect of the kinesin head-tail competition suggests it could explain the emergence of self-organized patterns inside cells, beyond regulation or cargo binding alone.

Age-related macular degeneration (AMD), the leading cause of blindness in the elderly, is characterized by progressive degeneration of retinal photoreceptors. Traditional disease models suggest that defective repression of thioester protein C3 activity by complement factor H (CFH) is a major contributor to pathogenesis in AMD and a related disease, early-onset drusen maculopathy (EODM). Our previous study identified novel functions for human CFH and Caenorhabditis elegans CFH-1 in the maintenance of inversin compartment integrity in photoreceptors and mechanosensory neurons, indicating that CFH has a novel, evolutionarily conserved role in cilia compartment organization that is distinct from its established function in alternative complement pathway regulation. Here, we investigate the C. elegans thioester protein TEP-1, an ancestral relative of C3 and other members of the AMCOM family (C4, C5, CD109, and alpha-2-macroglobulin). TEP-1 localizes to select glial cell surfaces and regulates inversin compartment organization and intraflagellar transport (IFT) within the cilia of ensheathed sensory neurons. In addition to revealing a novel role for an AMCOM family member in sensory neuron structure and protein transport, the localization of C3 and CFH on human photoreceptors provides support for noncanonical models of AMD and EODM pathogenesis in which defects in cilia structure and protein transport contribute directly to the progressive photoreceptor dysfunction that characterizes these diseases.

In response to external mechanical stimuli, cells remodel their actin cytoskeleton. Solo, a Rho guanine nucleotide exchange factor (RhoGEF), is involved in mechanical stress responses via cell-substrate adhesions. Using BioID, we identified PDZ-RhoGEF (PRG), a member of the RGS-RhoGEF (regulator of G protein signaling domain-containing RhoGEFs) family, as a Solo-interacting protein. Moreover, we found that Solo regulates PRG during the mechanical stress response. Furthermore, we identified leukemia-associated RhoGEF (LARG), another RGS-RhoGEF member, as a Solo-interacting protein; however, the functional role of this interaction remains unknown. Therefore, in this study, we investigated the interaction between Solo and LARG and found that LARG localizes to Solo accumulation sites at the basal plane and that LARG is required for Solo-induced actin polymerization. Additionally, Solo is required to maintain LARG activity in cells, and this interaction is related to actin regulation in response to substrate stiffness. We further investigated the relationship between LARG and PRG as a function of Solo. We noted that the double knockdown of PRG and LARG suppressed Solo-induced actin polymerization to the same extent or more as each single knockdown, indicating that these signaling pathways cooperatively regulate Solo-induced actin polymerization. [Media: see text].