Axonal autophagosomes use the ride-on service for retrograde transport toward the soma

Degradation of autophagic vacuoles (AVs) via lysosomes is an important homeostatic process in cells. Neurons are highly polarized cells with long axons, thus facing special challenges to transport AVs generated at distal processes toward the soma where mature acidic lysosomes are relatively enriched. We recently revealed a new motor-adaptor sharing mechanism driving autophagosome transport to the soma. Late endosome (LE)-loaded dynein-SNAPIN motor-adaptor complexes mediate the retrograde transport of autophagosomes upon their fusion with LEs in distal axons. This motor-adaptor sharing mechanism enables neurons to maintain effective autophagic clearance in the soma, thus reducing autophagic stress in axons. Therefore, our study reveals a new cellular mechanism underlying the removal of distal AVs engulfing aggregated misfolded proteins and dysfunctional organelles associated with several major neurodegenerative diseases.     

When trafficking and signaling mix: How subcellular location shapes G protein‐coupled receptor activation of heterotrimeric G proteins

G protein‐coupled receptors (GPCRs) physically connect extracellular information with intracellular signal propagation. Membrane trafficking plays a supportive role by “bookending” signaling events: movement through the secretory pathway delivers GPCRs to the cell surface where receptors can sample the extracellular environment, while endocytosis and endolysosomal membrane trafficking provide a versatile system to titrate cellular signaling potential and maintain homeostatic control. Recent evidence suggests that, in addition to these important effects, GPCR trafficking actively shapes the cellular signaling response by altering the location and timing of specific receptor‐mediated signaling reactions. Here, we review key experimental evidence underlying this expanding view, focused on GPCR signaling mediated through activation of heterotrimeric G proteins located in the cytoplasm. We then discuss lingering and emerging questions regarding the interface between GPCR signaling and trafficking.      

Staying in touch with the endocytic network: The importance of contacts for cholesterol transport

Cholesterol homeostasis is critical for cell function and human health. Cholesterol is heterogeneously distributed among cellular membranes, with the redistribution of endocytosed dietary cholesterol playing a pivotal role in the regulation of cholesterol homeostasis. While gaps remain in our understanding of intracellular dietary cholesterol transport, a highly complex network of pathways is starting to emerge, often involving inter‐dependent vesicular and non‐vesicular transport mechanisms. The last decade has seen a surge in interest in non‐vesicular transport and inter‐organellar communication at membrane contact sites. By providing platforms for protein interactions, signalling events, lipid exchange and calcium flux, membrane contact sites (MCS) are now appreciated as controlling the fate of large amounts of lipid and play central roles in the regulation and co‐ordination of endocytic trafficking. Here, we review the role of MCS in multiple pathways for cholesterol export from the endocytic pathway and highlight the intriguing interplay between vesicular and non‐vesicular transport mechanisms and relationship with neurodegenerative disease.     

Critical role of CAV1/caveolin-1 in cell stress responses in human breast cancer cells via modulation of lysosomal function and autophagy

CAV1 (caveolin 1, caveolae protein, 22kDa) is well known as a principal scaffolding protein of caveolae, a specialized plasma membrane structure. Relatively, the caveolae-independent function of CAV1 is less studied. Autophagy is a process known to involve various membrane structures, including autophagosomes, lysosomes, and autolysosomes for degradation of intracellular proteins and organelles. Currently, the function of CAV1 in autophagy remains largely elusive. In this study, we demonstrate for the first time that CAV1 deficiency promotes both basal and inducible autophagy. Interestingly, the promoting effect was found mainly in the late stage of autophagy via enhancing lysosomal function and autophagosome-lysosome fusion. Notably, the regulatory function of CAV1 in lysosome and autophagy was found to be caveolae-independent, and acts through lipid rafts. Furthermore, the elevated autophagy level induced by CAV1 deficiency serves as a cell survival mechanism under starvation. Importantly, downregulation of CAV1 and enhanced autophagy level were observed in human breast cancer cells and tissues. Taken together, our data reveal a novel function of CAV1 and lipid rafts in breast cancer development via modulation of lysosomal function and autophagy.     

Over‐expression of an inactive mutant cathepsin D increases endogenous alpha‐synuclein and cathepsin B activity in SH‐SY5Y cells

Parkinson's disease is a neurodegenerative movement disorder. The histopathology of Parkinson's disease comprises proteinaceous inclusions known as Lewy bodies, which contains aggregated α‐synuclein. Cathepsin D (CD) is a lysosomal protease previously demonstrated to cleave α‐synuclein and decrease its toxicity in both cell lines and mouse brains in vivo. Here, we show that pharmacological inhibition of CD, or introduction of catalytically inactive mutant CD, resulted in decreased CD activity and increased cathepsin B activity, suggesting a possible compensatory response to inhibition of CD activity. However, this increased cathepsin B activity was not sufficient to maintain α‐synuclein degradation, as evidenced by the accumulation of endogenous α‐synuclein. Interestingly, the levels of LC3, LAMP1, and LAMP2, proteins involved in autophagy‐lysosomal activities, as well as total lysosomal mass as assessed by LysoTracker flow cytometry, were unchanged. Neither autophagic flux nor proteasomal activities differs between cells over‐expressing wild‐type versus mutant CD. These observations point to a critical regulatory role for that endogenous CD activity in dopaminergic cells in α‐synuclein homeostasis which cannot be compensated for by increased Cathepsin B. These data support the potential need to enhance CD function in order to attenuate α‐synuclein accumulation as a therapeutic strategy against development of synucleinopathy.

     

A novel strategy to visualize vesicle‐bound kinesins reveals the diversity of kinesin‐mediated transport

In mammals, 15 to 20 kinesins are thought to mediate vesicle transport. Little is known about the identity of vesicles moved by each kinesin or the functional significance of such diversity. To characterize the transport mediated by different kinesins, we developed a novel strategy to visualize vesicle‐bound kinesins in living cells. We applied this method to cultured neurons and systematically determined the localization and transport parameters of vesicles labeled by different members of the Kinesin‐1, ‐2, and ‐3 families. We observed vesicle labeling with nearly all kinesins. Only six kinesins bound vesicles that undergo long‐range transport in neurons. Of these, three had an axonal bias (KIF5B, KIF5C and KIF13B), two were unbiased (KIF1A and KIF1Bβ), and one transported only in dendrites (KIF13A). Overall, the trafficking of vesicle‐bound kinesins to axons or dendrites did not correspond to their motor domain preference, suggesting that on‐vesicle regulation is crucial for kinesin targeting. Surprisingly, several kinesins were associated with populations of somatodendritic vesicles that underwent little long‐range transport. This assay should be broadly applicable for investigating kinesin function in many cell types.     

Regulation of endosomal clathrin and retromer‐mediated endosome to Golgi retrograde transport by the J‐domain protein RME‐8

After endocytosis, most cargo enters the pleiomorphic early endosomes in which sorting occurs. As endosomes mature, transmembrane cargo can be sequestered into inwardly budding vesicles for degradation, or can exit the endosome in membrane tubules for recycling to the plasma membrane, the recycling endosome, or the Golgi apparatus. Endosome to Golgi transport requires the retromer complex. Without retromer, recycling cargo such as the MIG‐14/Wntless protein aberrantly enters the degradative pathway and is depleted from the Golgi. Endosome‐associated clathrin also affects the recycling of retrograde cargo and has been shown to function in the formation of endosomal subdomains. Here, we find that the Caemorhabditis elegans endosomal J‐domain protein RME‐8 associates with the retromer component SNX‐1. Loss of SNX‐1, RME‐8, or the clathrin chaperone Hsc70/HSP‐1 leads to over‐accumulation of endosomal clathrin, reduced clathrin dynamics, and missorting of MIG‐14 to the lysosome. Our results indicate a mechanism, whereby retromer can regulate endosomal clathrin dynamics through RME‐8 and Hsc70, promoting the sorting of recycling cargo into the retrograde pathway.     

The role of intraorganellar Ca(2+) in late endosome-lysosome heterotypic fusion and in the reformation of lysosomes from hybrid organelles

We have investigated the requirement for Ca(2+) in the fusion and content mixing of rat hepatocyte late endosomes and lysosomes in a cell-free system. Fusion to form hybrid organelles was inhibited by 1,2-bis(2-aminophenoxy) ethane-N,N,N',N'-tetraacetic acid (BAPTA), but not by EGTA, and this inhibition was reversed by adding additional Ca(2+). Fusion was also inhibited by methyl ester of EGTA (EGTA-AM), a membrane permeable, hydrolyzable ester of EGTA, and pretreatment of organelles with EGTA-AM showed that the chelation of lumenal Ca(2+) reduced the amount of fusion. The requirement for Ca(2+) for fusion was a later event than the requirement for a rab protein since the system became resistant to inhibition by GDP dissociation inhibitor at earlier times than it became resistant to BAPTA. We have developed a cell-free assay to study the reformation of lysosomes from late endosome-lysosome hybrid organelles that were isolated from the rat liver. The recovery of electron dense lysosomes was shown to require ATP and was inhibited by bafilomycin and EGTA-AM. The data support a model in which endocytosed Ca(2+) plays a role in the fusion of late endosomes and lysosomes, the reformation of lysosomes, and the dynamic equilibrium of organelles in the late endocytic pathway.     

Routes and mechanisms of post‐endosomal cholesterol trafficking: A story that never ends

Mammalian cells acquire most exogenous cholesterol through receptor‐mediated endocytosis of low‐density lipoproteins (LDLs). After internalization, LDL cholesteryl esters are hydrolyzed to release free cholesterol, which then translocates to late endosomes (LEs)/lysosomes (LYs) and incorporates into the membranes by co‐ordinated actions of Niemann‐Pick type C (NPC) 1 and NPC2 proteins. However, how cholesterol exits LEs/LYs and moves to other organelles remain largely unclear. Growing evidence has suggested that nonvesicular transport is critically involved in the post‐endosomal cholesterol trafficking. Numerous sterol‐transfer proteins (STPs) have been identified to mediate directional cholesterol transfer at membrane contact sites (MCSs) formed between 2 closely apposed organelles. In addition, a recent study reveals that lysosome‐peroxisome membrane contact (LPMC) established by a non‐STP synaptotagmin VII and a specific phospholipid phosphatidylinositol 4,5‐bisphosphate also serves as a novel and important path for LDL‐cholesterol trafficking. These findings highlight an essential role of MCSs in intracellular cholesterol transport, and further work is needed to unveil how various routes are regulated and integrated to maintain proper cholesterol distribution and homeostasis in eukaryotic cells.      

A hot water extract of Aralia cordata activates bone marrow‐derived macrophages via a myeloid differentiation protein 88‐dependent pathway and protects mice from bacterial infection

In traditional Asian medicine, Aralia cordata (AC) is a known as a pain reliever and anti‐inflammatory drug. Although several of its biological activities have been reported, the immunomodulatory effects of a hot water extract of AC (HAC) have not yet been described. The aim of this study was to investigate whether HAC modulates the activation of macrophages, which play important roles in innate immune responses against microbial pathogens, and if so, to determine the molecular mechanisms by which HAC mediates this process. It was found that HAC activates bone marrow‐derived macrophages (BMDM) and increases amounts of nitric oxide and proinflammatory cytokines in a dose‐dependent manner. In addition, HAC was found to induce phosphorylation of NF‐κB and mitogen‐activated protein kinases (MAPKs), including c‐Jun N‐terminal kinases, extracellular signal‐regulated kinases and p38. Interestingly, these effects were absent in BMDM prepared from myeloid differentiation protein 88‐knockout mice. Polysaccharides from HAC exerted stronger immunostimulatory effects than HAC itself. Furthermore, orally administered HAC clearly enhanced clearance of the intracellular pathogen Listeria monocytogenes by boosting innate immune responses. These results demonstrate that HAC exerts immunostimulatory effects through the TLR/MyD88 and NF‐κB/MAPK signal transduction pathways.     

A putative Toll/interleukin‐1 receptor domain protein from Helicobacter pylori is dimeric in solution and interacts with human Toll‐like receptor adaptor myeloid differentiation primary response 88

Helicobacter pylori, an important human pathogen, is capable of causing persistent infection with minimal immune response. The first line of defense during H. pylori infection is through gastric epithelial cells that present TLR, A family of bacterial proteins that share homology with the Toll/IL‐1 receptor (TIR) domain were identified. Bacterial TIR proteins (BTP) mimic human TIR domain proteins and act on myeloid differentiation primary response gene 88 (MyD88) signaling pathways to suppress TLR signaling. H. pylori may also produce a similar protein. A putative H. pylori BTP was found based on sequence homology. The corresponding gene hp1437 was inserted into an expression vector in fusion with an N‐terminal cleavable 6his‐tag. The recombinant protein, 6his‐HP1437, was purified using nickel affinity chromatography with a yield of 8 mg/L culture. Oligomerization of HP1437 was investigated by size‐exclusion chromatography. It was found that HP1437 forms dimers in solution similar to other BTPs. Furthermore, glutathione S‐transferase pull down assays identified an interaction between HP1437 and human TIR domain adaptor MyD88. These findings suggest that HP1437 has the characteristic features of BTPs and may play a direct role in reducing immune response against H. pylori by binding to MyD88 and pave the way for an in‐depth characterization of this putative novel H. pylori virulence factor.     

Rab12 regulates mTORC1 activity and autophagy through controlling the degradation of amino‐acid transporter PAT4

Autophagy is an evolutionarily conserved catabolic mechanism that targets intracellular molecules and damaged organelles to lysosomes. Autophagy is achieved by a series of membrane trafficking events, but their regulatory mechanisms are poorly understood. Here, we report small GTPase Rab12 as a new type of autophagic regulator that controls the degradation of an amino‐acid transporter. Knockdown of Rab12 results in inhibition of autophagy and in increased activity of mTORC1 (mammalian/mechanistic target of rapamycin complex 1), an upstream regulator of autophagy. We also found that Rab12 promotes constitutive degradation of PAT4 (proton‐coupled amino‐acid transporter 4), whose accumulation in Rab12‐knockdown cells modulates mTORC1 activity and autophagy. Our findings reveal a new mechanism of regulation of mTORC1 signalling and autophagy, that is, quality control of PAT4 by Rab12.     

Iejimalides A and B inhibit lysosomal vacuolar H+‐ATPase (V‐ATPase) activity and induce S‐phase arrest and apoptosis in MCF‐7 cells

Iejimalides are novel macrolides that are cytostatic or cytotoxic against a wide range of cancer cells at low nanomolar concentrations. A recent study by our laboratory characterized the expression of genes and proteins that determine the downstream effects of iejimalide B. However, little is known about the cellular target(s) of iejimalide or downstream signaling that lead to cell‐cycle arrest and/or apoptosis. Iejimalides have been shown to inhibit the activity of vacuolar H⁺‐ATPase (V‐ATPase) in osteoclasts, but how this inhibition may lead to cell‐cycle arrest and/or apoptosis in epithelial cells is not known. In this study, MCF‐7 breast cancer cells were treated with iejimalide A or B and analyzed for changes in cell‐cycle dynamics, apoptosis, lysosomal pH, cytoplasmic pH, mitochondrial membrane potential, and generation of reactive oxygen species. Both iejimalides A and B sequentially neutralize the pH of lysosomes, induce S‐phase cell‐cycle arrest, and trigger apoptosis in MCF‐7 cells. Apoptosis occurs through a mechanism that involves oxidative stress and mitochondrial depolarization but not cytoplasmic acidification. These data confirm that iejimalides inhibit V‐ATPase activity in the context of epithelial tumor cells, and that this inhibition may lead to a lysosome‐initiated cell death process. J. Cell. Biochem. 109: 634–642, 2010. © 2009 Wiley‐Liss, Inc.     

Distinct complexes of yeast Snx4 family SNX‐BARs mediate retrograde trafficking of Snc1 and Atg27

The yeast SNX4 sub‐family of sorting nexin containing a Bin‐Amphiphysin‐Rvs domain (SNX‐BAR) proteins, Snx4/Atg24, Snx41 and Atg20/Snx42, are required for endocytic recycling and selective autophagy. Here, we show that Snx4 forms 2 functionally distinct heterodimers: Snx4‐Atg20 and Snx4‐Snx41. Each heterodimer coats an endosome‐derived tubule that mediates retrograde sorting of distinct cargo; the v‐SNARE, Snc1, is a cargo of the Snx4‐Atg20 pathway, and Snx4‐Snx41 mediates retrograde sorting of Atg27, an integral membrane protein implicated in selective autophagy. Live cell imaging of individual endosomes shows that Snx4 and the Vps5‐Vps17 retromer SNX‐BAR heterodimer operate concurrently on a maturing endosome. Consistent with this, the yeast dynamin family protein, Vps1, which was previously shown to promote fission of retromer‐coated tubules, promotes fission of Snx4‐Atg20 coated tubules. The results indicate that the yeast SNX‐BAR proteins coat 3 distinct types of endosome‐derived carriers that mediate endosome‐to‐Golgi retrograde trafficking.      

A negative feedback regulation of MTORC1 activity by the lysosomal Ca2+ channel MCOLN1 (mucolipin 1) using a CALM (calmodulin)-dependent mechanism

Macroautophagy/autophagy is an evolutionarily conserved pathway that is required for cellular homeostasis, growth and survival. The lysosome plays an essential role in autophagy regulation. For example, the activity of MTORC1, a master regulator of autophagy, is regulated by nutrients within the lysosome. Starvation inhibits MTORC1 causing autophagy induction. Given that MTORC1 is critical for protein synthesis and cellular homeostasis, a feedback regulatory mechanism must exist to restore MTORC1 during starvation. However, the molecular mechanism underlying this feedback regulation is unclear. In this study, we report that starvation activates the lysosomal Ca²⁺ release channel MCOLN1 (mucolipin 1) by relieving MTORC1's inhibition of the channel. Activated MCOLN1 in turn facilitates MTORC1 activity that requires CALM (calmodulin). Moreover, both MCOLN1 and CALM are necessary for MTORC1 reactivation during prolonged starvation. Our data suggest that lysosomal Ca²⁺ signaling is an essential component of the canonical MTORC1-dependent autophagy pathway and MCOLN1 provides a negative feedback regulation of MTORC1 to prevent excessive loss of MTORC1 function during starvation. The feedback regulation may be important for maintaining cellular homeostasis during starvation, as well as many other stressful or disease conditions.