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In recent years, cell biologists have uncovered a number of new functions for proteins that were previously thought to operate solely in membrane trafficking. These alternative roles, termed moonlighting functions, can occur at distinct intracellular sites or at different stages of the cell cycle. Here, I evaluate the evidence for mitotic moonlighting functions of proteins that have membrane trafficking roles during interphase. The aim is to identify key issues facing the field and to outline important questions for future work....
Unconventional secretory proteins represent a subpopulation of extracellular factors that are exported from eukaryotic cells by mechanisms that do not depend on the endoplasmic reticulum and the Golgi complex. Various pathways have been implicated in unconventional secretion including those involving intracellular membrane‐bound intermediates and others that are based on direct protein translocation across plasma membranes. Interleukin 1β (IL1β) and fibroblast growth factor 2 (FGF2) are classical examples of unconventional secretory proteins with IL1β believed to be present in intracellular vesicles prior to secretion. By contrast, FGF2 represents an example of a non‐vesicular mechanism of unconventional secretion. Here, the author discusses the current knowledge about the molecular machinery being involved in FGF2 secretion. To reveal both differential and common requirements, this review further aims at a comprehensive comparison of this mechanism with other unconventional secretory processes. In particular, a potentially general role of tyrosine phosphorylation as a regulatory signal in unconventional protein secretion will be discussed....
Osteoclasts are specialized cells that secrete lysosomal acid hydrolases at the site of bone resorption, a process critical for skeletal formation and remodeling. However, the cellular mechanism underlying this secretion and the organization of the endo‐lysosomal system of osteoclasts have remained unclear. We report that osteoclasts differentiated in vitro from murine bone marrow macrophages contain...
Membrane remodeling is an important aspect in organelle biogenesis. We show that different peroxisome membrane proteins that play a role in organelle biogenesis and proliferation (Pex8, Pex10, Pex14, Pex25 and Pex11) are subject to spatiotemporal behavior during organelle development. Using fluorescence microscopy analysis of Hansenula polymorpha dnm1 cells that are blocked in the normal fission process,...
Immuno‐transmission electron microscopy (TEM) is the technique of choice for high‐resolution localization of proteins in fixed specimen. Here we introduce 2 novel methods for the fixation of sections from cryo‐immobilized samples that result in excellent ultrastructural preservation. These high‐speed fixation techniques, both called VIS2FIX, allow for a reduction in sample preparation time from at least 1 week to only 8 h. The methods were validated in immuno‐TEM experiments on THP‐1 monocytes, human umbilical vein endothelial cells (HUVECs) and Madin–Darby canine kidney (MDCK‐II) cells. The fixation and retention of neutral lipids is demonstrated, offering unique prospects for the application of immuno‐TEM in the lipidomics field. Furthermore, the VIS2FIX methods were successfully employed in correlative fluorescence and electron microscopy....
Microglia are immune effector cells in the central nervous system (CNS) and their activation, migration and proliferation play crucial roles in brain injuries and diseases. We examined the role of intracellular Ca2+‐independent phospholipase A2 (iPLA2) in the regulation of microglia chemotaxis toward ADP. Inhibition of iPLA2 by 4‐bromoenol lactone (BEL) or iPLA2 knockdown exerted a significant inhibition...
A well‐orchestrated hierarchy of molecular events is required for successful initiation and maturation of clathrin‐coated pits (CCPs). Nevertheless, CCPs display a broad range of lifetimes. This dynamic heterogeneity could either reflect differences in the temporal hierarchy of molecular events, or similar CCP maturation processes with variable kinetics. To address this question, we have used multi‐channel...
Modification of histones is critical for the regulation of all chromatin‐templated processes. Yeast Rtt109 is a histone acetyltransferase (HAT) that acetylates H3 lysines 9, 27 and 56. Rtt109 associates with and is stabilized by Nap1 family histone chaperone Vps75. Our data suggest Vps75 and Nap1 have some overlapping functions despite their different cellular localization and histone binding specificity...
Like other apicomplexan parasites, Toxoplasma gondii actively invades host cells using a combination of secretory proteins and an acto‐myosin motor system. Micronemes are the first set of proteins secreted during invasion that play an essential role in host cell entry. Many microneme proteins (MICs) function in protein complexes, and each complex contains at least one protein that displays a cleavable...
Chlamydiae are Gram negative, obligate intracellular bacteria, and Chlamydia trachomatis is the etiologic agent of the most commonly reported sexually transmitted disease in the United States. Chlamydiae undergo a biphasic life cycle that takes place inside a parasitophorous vacuole termed an inclusion. Chlamydial infections have been epidemiologically linked to cervical cancer in patients previously...
SNARE proteins are essential fusion mediators for many intracellular trafficking events. Here, we investigate the role of Syntaxin7 (Stx7) in the release of lytic granules from cytotoxic T lymphocytes (CTLs). We show that Stx7 is expressed in CTLs and is preferentially localized to the region of lytic granule release, the immunological synapse (IS). Interference of Stx7 function by expression of a dominant‐negative Stx7 construct or by small interfering RNA leads to a dramatic reduction of CTL‐mediated killing of target cells. Real‐time visualization of individual lytic granules at the IS by evanescent wave microscopy reveals that lytic granules in Stx7‐deprived CTLs not only fail to fuse with the plasma membrane but even fail to accumulate at the IS. Surprisingly, the accumulation defect is not caused by an overall reduction in lytic granule number, but by a defect in the trafficking of T cell receptors (TCRs) through endosomes. Subsequent high‐resolution nanoscopy shows that Stx7 colocalizes with Rab7 on late endosomes. We conclude from these data that the accumulation of recycling TCRs at the IS is a SNARE‐dependent process and that Stx7‐mediated processing of recycling TCRs through endosomes is a prerequisite for the cytolytic function of CTLs....
The human immunodeficiency virus 1 (HIV‐1) Nef protein is a pathogenicity factor required for effective progression to AIDS, which modulates host cell signaling pathways and T‐cell receptor internalization. We have determined the crystal structure of Nef, allele SF2, in complex with an engineered SH3 domain of human Hck showing unnaturally tight binding and inhibitory potential toward Nef. This complex provides the most complete Nef structure described today, and explains the structural basis of the high affinity of this interaction. Intriguingly, the 33‐residue C‐terminal flexible loop is resolved in the structure by its interactions with a highly conserved hydrophobic groove on the core domain of an adjacent Nef molecule. The loop mediates the interaction of Nef with the cellular adaptor protein machinery for the stimulated internalization of surface receptors. The endocytic dileucine‐based sorting motif is exposed at the tip of the acidic loop, giving the myristoylated Nef protein a distinctly dipolar character. The intermolecular domain assembly of Nef provides insights into a possible regulation mechanism for cargo trafficking....
Cytotoxic T lymphocytes (CTL) are potent killers of virally infected and tumorigenic cells. Upon recognition of target cells, CTL undergo polarized secretion of secretory lysosomes at the immunological synapse (IS) that forms between CTL and target. However, the molecular machinery involved in the polarization of secretory lysosomes is still largely uncharacterized. In this paper, we investigated the role of Rab7 in the polarization of secretory lysosomes. We show that silencing of Rab7 by RNA interference reduces the ability of CTL to kill targets. GTP‐bound Rab7 and Rab interacting lysosomal protein, RILP, interact and both localize to secretory lysosomes in CTL. Over‐expression of RILP recruits dynein to the membranes of secretory lysosomes and triggers their movement toward the centrosome. Together, these results suggest that Rab7 may play a role in secretory lysosome movement toward the centrosome by interacting with RILP to recruit the minus‐end motor, dynein....
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