Journal of cell science最新文献

Although many cancer cells proliferate by metabolizing extracellular proteins internalized by macropinocytosis and degraded in lysosomes, the extent to which macropinocytosis contributes to the growth of other metazoan cells remains undefined. This study analyzed macropinocytosis in proliferating murine macrophages as a mechanism for extracting amino acids from growth media. Macrophages internalized the fluid-phase probe Lucifer Yellow by macropinocytosis and recycled much of it from their lysosomes by a first-order process. Inhibitors of pinocytosis inhibited cell growth. Removal of the essential amino acid leucine from growth medium reduced proliferation, and allowed analysis of pinocytosis and the higher growth rates achieved by supplementation with either free leucine or bovine serum albumin (BSA) as a source of leucine. Macrophages could proliferate by utilizing macropinocytosis and digestion of BSA. In contrast, growth on free leucine exceeded the capacity of macropinocytosis to extract leucine from the medium. Dye molecules released from proteins by hydrolysis in lysosomes were recycled from cells efficiently. We propose that macropinocytosis concentrates large solutes, such as proteins, into lysosomes but allows amino acids and other products of lysosomal hydrolases to redistribute into macropinosomes and outside of the cell.

Neurotransmitter release is triggered rapidly by Ca2+ binding to synaptotagmin-1 in cooperation with the soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs). Synaptotagmin-1 is believed to facilitate membrane fusion by inserting the Ca2+-binding loops of its two C2 domains into membranes, thus perturbing the bilayers and/or inducing curvature. However, this direct role has been questioned by the observation that when the synaptotagmin-1 C2B domain binds the SNARE complex, its Ca2+-binding loops point away from the fusion site. Recent data together with older results suggested a natural explanation for this paradox. Molecular dynamics simulations indicate that placing the Ca2+-binding loops close to the fusion site hinders SNARE-mediated membrane fusion. Electron paramagnetic resonance, nuclear magnetic resonance and fluorescence spectroscopy show that, upon Ca2+ binding, the C2B domain reorients on the membrane and can partially dissociate from the membrane-anchored SNARE complex. Electrophysiological studies strongly suggest that such reorientation of the C2B domain with respect to the SNAREs is crucial for neurotransmitter release. In this Hypothesis article, we discuss how these findings have led to a model whereby Ca2+-induced reorientation the C2B domain causes synaptotagmin-1 to act remotely as a lever, pulling the SNARE complex and facilitating SNARE conformational changes that trigger fast membrane fusion.

Polytopic copper (Cu)-ATPases are central regulators of the essential micronutrient copper in all organisms. In polarized epithelia, the vertebrate homologues ATP7A and ATP7B undergo copper-induced trafficking from the trans-Golgi network (TGN) to basolateral and apical membranes, respectively, to mediate efflux of excess copper. To probe (1) inter-domain interactions that drive trafficking and (2) the extent of divergence between homologous domains constituting Cu-ATPases, we replaced the copper-binding N-terminal (NT), nucleotide-binding (NBD) and/or C-terminal (CT) domains of ATP7B with those of ATP7A. The functionally active chimeras exhibited distinct trafficking phenotypes. Notably, the ATP7B-NT substitution led to constitutive basolateral membrane trafficking, whereas simultaneous NT-NBD substitution led to steady-state TGN localization, suggesting that interaction between the two domains, as confirmed by in vitro NT-NBD-binding studies, might be essential for TGN localization. Interestingly, reciprocal replacement of the ATP7A-NBD and -NT with that from ATP7B did not rescue membrane localization, indicating that domain compatibility is restricted, suggesting greater evolutionary divergence of ATP7B domains. Analysing orthologous Cu-ATPase domain-sequences from diverse organisms, however, revealed similar evolutionary relationships between the NT and NBD, suggesting their co-evolution. We thus correlate the copper-responsive trafficking ability of Cu-ATPases with evolutionary stringency imparted onto Cu-ATPase domains.

Sensory cilia have a complex bipartite architecture containing 9+0 connecting cilia at the inner segment and singlet microtubule-supported highly membranous outer segments essential for receptor display. The mechanisms underlying the formation of such highly branched morphology and its microtubule-rich ciliary cytoskeleton are unclear. Here, we show that individual olfactory cilium inside the large basiconic sensillum grows in episodic steps following several pulsatile influxes of tubulin in developing Drosophila antenna. Transient elevations of the microtubule end-binding protein EB1 precede the tubulin influx events. We also demonstrate that EB1 interacts explicitly with the cargo-binding tail domain of Drosophila KLP68D, a kinesin-2β orthologue. Loss of EB1 in olfactory neurons during the outer segment growth reduces the tubulin influx and affects cilia stability. Finally, we show that the EB1 and tubulin influxes into the distal outer segment of the olfactory cilia require kinesin-2. Altogether, our findings elucidate a role of active EB1 transport in promoting the growth and stability of long-lived metazoan cilia involved in sensory perception.

Integrin-mediated adhesion regulates cellular survival and mechanotransduction, processes often deregulated in cancers. During breast tumor progression, matrix stiffening influences cytoskeletal organization, although its effect on organelle organization and function remains unclear. Here, we examine how Golgi organization responds to matrix stiffness sensing in breast cancer cells. In adherent MDA-MB-231 cells, the Golgi becomes progressively more compact and organized with increasing matrix stiffness, accompanied by enhanced tubulin acetylation, indicating stiffness-dependent regulation. In contrast, MCF7 cells display a diffused or disorganized Golgi regardless of matrix stiffness. AXL, a receptor tyrosine kinase differentially expressed in MDA-MB-231 cells and absent in MCF7, localizes prominently to the Golgi. Inhibition or knockdown of AXL disrupted stiffness-dependent Golgi organization in MDA-MB-231 cells, whereas stable AXL expression in MCF7 restored Golgi organization at higher stiffness. A stiffness-dependent increase in AXL and Arf1 expression regulates Arf1 activation and localization to control mechanosensitive Golgi organization. Inhibition of AXL and/or Arf1 disrupted Golgi organization, tubulin acetylation and cell-surface glycosylation. Together, our findings reveal a mechanoresponsive AXL-Arf1-Golgi signaling axis that integrates matrix stiffness sensing with Golgi organization and function in breast cancer cells.

Mitochondrial dynamics are defined by the continuous processes of fusion and fission that regulate mitochondrial shape, distribution and activity. They are also involved in cellular functions of mitochondria, such as energy production, metabolic adaptation, apoptosis and cellular stress responses. Consequently, these organelle dynamics play a crucial role in development, growth, differentiation and disease. Mitochondrial morphology is controlled by Drp1 (also known as DNM1L) and Fis1, which drive fission, whereas Opa1, Mfn1 and Mfn2 mediate fusion. The transcription, activation and degradation of these proteins are often regulated by signaling cascades that are crucial for stem cell maintenance and differentiation. In turn, mitochondrial dynamics regulate key outcomes of these pathways. We explore the interplay between mitochondrial fusion and fission proteins and such signaling pathways, including Notch, receptor tyrosine kinase, JNK, Hippo and mTOR signaling, finding that stem cell renewal and differentiation states are dependent on the regulation of signaling pathways by mitochondrial morphology and activity. Overall, this Review highlights how mitochondrial morphology and activity crucially regulate stem cell division for renewal and differentiation, examining their impact across diverse systems.

Tip growth is closely tied to fungal pathogenicity. Budding yeast Spa2 (the homolog of GIT1 and GIT2 in mammals), a multi-domain protein and member of the polarisome, orchestrates tip growth in yeasts and other fungi. We identified a conserved short linear motif in the Rab GTPase-activating proteins (RabGAPs) Msb3 and Msb4, and the MAP kinase kinases Ste7 and Mkk1, which mediates their interaction with Spa2. AlphaFold predictions suggest that these initially unstructured motifs adopt an α-helical conformation upon binding to the hydrophobic cleft in the N-terminal domain of Spa2. Altering the predicted key contact residues in either Spa2 or the motif reduces complex stability. Such mutations also cause mis-localization of Msb3, Msb4 and Ste7 within the cell. Deleting the motif in Msb3 or Msb4 abolishes tip-directed growth of the yeast bud. Protein assemblies that spatially confine secretion to specific membrane regions are a common feature of eukaryotic cells. Accordingly, complexes between proteins with this motif and Spa2 were predicted in orthologs and paralogs across selected Opisthokonta, including pathogenic fungi and humans. A search for functional motifs in conformationally flexible regions of all yeast proteins identified Dse3 as a novel Spa2-binding partner.

Cell shape regulation is important for many biological processes. Some cell shape-regulating proteins harbor mechanoresponsive properties that enable them to sense and respond to mechanical cues. In Dictyostelium discoideum, mechanoresponsive network proteins formed by proteins such as myosin II, cortexillin I and IQGAP1 assemble in the cytoplasm into macromolecular complexes, which we term contractility kits (CKs). In our previous studies, we identified the RNA-binding protein RNP1A as a genetic interactor with the cytoskeletal machinery of the cell and as a biochemical interactor of cortexillin I, using in vivo fluorescence cross-correlation spectroscopy. In this study, we show that Dictyostelium rnp1A knockdown cells have reduced cell proliferation, reduced adhesion, defective cytokinesis, and a gene expression profile that indicates rnp1A knockdown cells shift away from the vegetative growth state. Some of the transcripts RNP1A binds encode proteins involved in macropinocytosis, a crucial cell shape change process. Loss of other CK proteins leads to macropinocytotic defects characterized by reduced macropinocytotic crown size. RNP1A interacts with IQGAP1, leading to crosstalk during macropinocytosis. Overall, RNP1A binds transcripts and contributes to cell mechanics and cell shape change processes through interactions with CK proteins.

A significant challenge in studying the biology of the Drosophila centriole is its small size. Advanced super-resolution techniques have provided valuable insights but require specialized equipment and can be difficult to implement in tissues. Expansion microscopy (ExM) offers an accessible alternative, yet its application in Drosophila centriole research has been sparse. We provide an ExM protocol for cultured S2 cells and fly tissues that reveals new insights into procentriole biology. In S2 cells we document overduplication in the form of the classic 'rosettes', while in spermatids we uncover an unexpected movement of the procentriole-like structure (PCL). ExM has also refined existing molecular models. In S2 cells we document the distal tip protein Cep97 as a ring, which clarifies its role in capping the growing centriole. In spermatids, we spatially segregate the inner nuclear membrane protein Spag4 and the cytoplasmic protein Yuri, leading to the new hypothesis that they play independent roles at the centriole-nucleus contact site. Finally, we show that our ExM protocol is a hypothesis generator and discovery tool applicable beyond Drosophila centrioles by imaging synaptonemal complexes in the Plodia interpunctella moth.

Our changing climate poses increasingly severe threats to human and environmental health. Scientific research is essential for understanding and mitigating these effects, but how can cell biologists support this goal? In this Essay, Journal of Cell Science has invited cell biologists from across disciplines and career stages to share their perspectives on how cell biology can address climate-related questions. Their research ranges from practical innovations to fundamental functional studies. How can we re-route metabolic pathways to reduce industrial emissions? What can plankton-microbe interactions tell us about the impact of marine pollution? How can an in-depth understanding of cellular processes help us design more resilient crops to address specific challenges faced in West African countries? Could developments in stem cell biology help safeguard biodiversity? What can we learn from the way deep-sea squid adapt to changing environments on the cellular level? These examples illustrate an increasing drive to apply broad insights and techniques from the world of cell biology to this urgent, global challenge.