Rab11-dependent Pathway Research Articles

RAB11-Mediated Trafficking and Human Cancers: An Updated Review.

Many disorders block and subvert basic cellular processes in order to boost their progression. One protein family that is prone to be altered in human cancers is the small GTPase RAB11 family, the master regulator of vesicular trafficking. RAB11 isoforms function as membrane organizers connecting the transport of cargoes towards the plasma membrane with the assembly of autophagic precursors and the generation of cellular protrusions. These processes dramatically impact normal cell physiology and their alteration significantly affects the survival, progression and metastatization as well as the accumulation of toxic materials of cancer cells. In this review, we discuss biological mechanisms ensuring cargo recognition and sorting through a RAB11-dependent pathway, a prerequisite to understand the effect of RAB11 alterations in human cancers.

Intermedin protects against sepsis by concurrently re-establishing the endothelial barrier and alleviating inflammatory responses

Sepsis is a life-threatening condition caused by dysregulated host responses to infection. Widespread vascular hyperpermeability and a “cytokine storm” are two pathophysiological hallmarks of sepsis. Here, we show that intermedin (IMD), a member of the calcitonin family, alleviates organ injury and decreases mortality in septic mice by concurrently alleviating vascular leakage and inflammatory responses. IMD promotes the relocation of vascular endothelial cadherin through a Rab11-dependent pathway to dynamically repair the disrupted endothelial junction. Additionally, IMD decreases inflammatory responses by reducing macrophage infiltration via downregulating CCR2 expression. IMD peptide administration ameliorates organ injuries and significantly improves the survival of septic mice, and the experimental results correlate with the clinical data. Patients with high IMD levels exhibit a lower risk of shock, lower severity scores, and greatly improved survival outcomes than those with low IMD levels. Based on our data, IMD may be an important self-protective factor in response to sepsis.

A Ca2+-stimulated exosome release pathway in cancer cells is regulated by Munc13-4.

Cancer cells secrete copious amounts of exosomes, and elevated intracellular Ca2+ is critical for tumor progression and metastasis, but the underlying cellular mechanisms are unknown. Munc13-4 is a Ca2+-dependent SNAP receptor- and Rab-binding protein required for Ca2+-dependent membrane fusion. Here we show that acute elevation of Ca2+ in cancer cells stimulated a fivefold increase in CD63+, CD9+, and ALIX+ exosome release that was eliminated by Munc13-4 knockdown and not restored by Ca2+ binding-deficient Munc13-4 mutants. Direct imaging of CD63-pHluorin exosome release confirmed its Munc13-4 dependence. Depletion of Munc13-4 in highly aggressive breast carcinoma MDA-MB-231 cells reduced the size of CD63+ multivesicular bodies (MVBs), indicating a role for Munc13-4 in MVB maturation. Munc13-4 used a Rab11-dependent trafficking pathway to generate MVBs competent for exosome release. Membrane type 1 matrix metalloproteinase trafficking to MVBs by a Rab11-dependent pathway was also Munc13-4 dependent, and Munc13-4 depletion reduced extracellular matrix degradation. These studies identify a novel Ca2+- and Munc13-4-dependent pathway that underlies increased exosome release by cancer cells.

Myosin Va mediates BDNF-induced postendocytic recycling of full-length TrkB and its translocation into dendritic spines.

Brain-derived neurotrophic factor (BDNF) plays an important role in neuronal survival, neurite outgrowth and synaptic plasticity by activating the receptor tropomyosin receptor kinase B (TrkB, also known as NTRK2). TrkB has been shown to undergo recycling after BDNF stimulation. We have previously reported that full-length TrkB (TrkB-FL) are recycled through a Rab11-dependent pathway upon BDNF stimuli, which is important for the translocation of TrkB-FL into dendritic spines and for the maintenance of prolonged BDNF downstream signaling during long-term potentiation (LTP). However, the identity of the motor protein that mediates the local transfer of recycled TrkB-FL back to the plasma membrane remains unclear. Here, we report that the F-actin-based motor protein myosin Va (Myo5a) mediates the postendocytic recycling of TrkB-FL. Blocking the interaction between Rab11 and Myo5a by use of a TAT-tagged peptide consisting of amino acids 55-66 of the Myo5a ExonE domain weakened the association between TrkB-FL and Myo5a and thus impaired TrkB-FL recycling and BDNF-induced TrkB-FL translocation into dendritic spines. Finally, inhibiting Myo5a-mediated TrkB-FL recycling led to a significant reduction in prolonged BDNF downstream signaling. Taken together, these results show that Myo5a mediates BDNF-dependent TrkB-FL recycling and contributes to BDNF-induced TrkB spine translocation and prolonged downstream signaling.

Morphological changes associated with the genesis and development of an excitatory glutemergic synapse: An integrated framework model

Morphological changes associated with the genesis and development of an excitatory glutemergic synapse: An integrated framework model

BDNF-Dependent Recycling Facilitates TrkB Translocation to Postsynaptic Density during LTP via a Rab11-Dependent Pathway

Brain-derived neurotrophic factor (BDNF) plays an important role in the activity-dependent regulation of synaptic structure and function via tropomyosin related kinase B (TrkB) receptor activation. However, whether BDNF could regulate TrkB levels at synapse during long-term potentiation (LTP) is still unknown. We show in cultured rat hippocampal neurons that chemical LTP (cLTP) stimuli selectively promote endocytic recycling of BDNF-dependent full-length TrkB (TrkB-FL) receptors, but not isoform T1 (TrkB.T1) receptors, via a Rab11-dependent pathway. Moreover, neuronal-activity-enhanced TrkB-FL recycling could facilitate receptor translocation to postsynaptic density and enhance BDNF-induced extracellular signal-regulated kinase and phosphatidylinositol 3-kinase activation and rat hippocampal neuron survival. Finally, we found that cLTP could stimulate the switch of Rab11 from an inactive to an active form and that GTP-bound Rab11 could enhance the interaction between TrkB-FL and PSD-95. Therefore, the recycling endosome could serve as a reserve pool to supply TrkB-FL receptors for LTP maintenance. These findings provide a mechanistic link between Rab11-dependent endocytic recycling and TrkB modulation of synaptic plasticity.

The armadillo protein p0071 is involved in Rab11-dependent recycling

p0071 is an intercellular junction protein of the p120 catenin family. We have identified Rab11a as a novel interaction partner of p0071. p0071 interacted preferentially with active Rab11a. Knockdown experiments revealed an interdependent regulation of both proteins. On the one hand, p0071 depletion induced a perinuclear accumulation of Rab11, suggesting a role of p0071 in the anterograde transport of Rab11 from the pericentrosomal region to the plasma membrane but not in retrograde transport. p0071 as well as Rab11 depletion increased transferrin receptor recycling indicating that p0071-induced Rab11 mislocalization interfered with Rab11 function and shifted recycling from the slow Rab11-dependent pathway to the fast Rab4-dependent pathway. When p0071 or Rab11 depletion was combined with a Rab4 knockdown the effect was reversed. On the other hand, Rab11a depletion increased p0071 recycling to cell contacts thereby identifying p0071 as a Rab11 cargo protein. This correlated with increased intercellular adhesion. Thus, we propose that p0071 has a key role in regulating recycling through the Rab11-dependent perinuclear recycling compartment, and links the regulation of adherens junctions to recycling to allow dynamic modulation of intercellular adhesion.

Cannabinoid receptor 2 undergoes Rab5-mediated internalization and recycles via a Rab11-dependent pathway

Cannabinoid receptor 2 (CB2) is a GPCR highly expressed on the surface of cells of the immune system, supporting its role in immunomodulation. This study has investigated the trafficking properties of this receptor when stably expressed by HEK-293 cells. As previously reported, cell surface CB2 rapidly internalized upon exposure to agonist. Direct evidence of CB2 recycling was observed upon competitive removal of the stimulating agonist by inverse agonist. CB2 also underwent slow constitutive internalization when agonist was absent and was up-regulated in the presence of inverse agonist. Co-expression of CB2 and dominant negative Rab5 resulted in a significantly reduced capacity for receptors to internalize with no effect on recycling of the internalized receptors. Conversely, co-expression with dominant negative Rab11 did not alter the ability of CB2 to internalize but did impair their ability to return to the cell surface. Co-expression of wild-type, dominant negative or constitutively active Rab4 with CB2 did not alter basal surface expression, extent of internalization, or extent of recycling. These results suggest that Rab5 is involved in CB2 endocytosis and that internalized receptors are recycled via a Rab11 associated pathway rather than the rapid Rab4 associated pathway. This report provides the first comprehensive description of CB2 internalization and recycling to date.

The internalization of the M 2 and M 4 muscarinic acetylcholine receptors involves distinct subsets of small G-proteins

Multiple mechanisms exist for the endocytosis of receptors from the cell surface. While the M 1, M 3, and M 4 subtypes of muscarinic acetylcholine receptors internalize through the well-characterized mechanism of clathrin coated vesicles, the mechanism of M 2 endocytosis is not well defined. Because the M 2 and M 4 receptors transduce their signals through the same second messengers but internalize though different pathways, we tested the ability of several small G-proteins to regulate the agonist-induced endocytosis of M 2 and M 4 in JEG-3 human choriocarcinoma cells. Dominant-negative Rab5 as well as both wild-type and dominant-negative Rab11 inhibited M 4 but not M 2 endocytosis. In contrast, a dominant-negative Arf6 as well as wild-type Rab22 increased M 2 but not M 4 endocytosis. We used immunocytochemistry to show that in unstimulated cells, the M 2 and M 4 receptors co-localize on the cell surface, whereas after stimulation M 2 and M 4 are in distinct vesicular compartments. In this study, we demonstrate that agonist-induced internalization of the M 2 receptor utilizes an Arf6, Rab22 dependent pathway, while the M 4 receptor undergoes agonist-induced internalization through a Rab5, Rab11 dependent pathway. Additionally, we show that Rab15 and RhoA are not involved in either pathway in JEG-3 cells.

ATP depletion inhibits the endocytosis of ClC‐2

The chloride channel, ClC-2 is expressed ubiquitously and participates in multiple physiological processes. In particular, ClC-2 has been implicated in the regulation of neuronal chloride ion homeostasis and mutations in ClC-2 are associated with idiopathic generalized epilepsy. Despite the physiological and pathophysiological significance of this channel, its regulation remains incompletely understood. The functional expression of ClC-2 at the cell surface has been shown to be enhanced by depletion of cellular ATP, implicating its possible role in cellular energy sensing. In the present study, biochemical assays of cell surface expression suggest that this gain of function reflects, in part, an increase in channel number due to the reduction in ClC-2 internalization by endocytosis. Cell surface expression of the disease-causing mutant: G715E, thought to lack wild-type nucleotide binding affinity, is similarly affected, suggesting that ATP-depletion modifies the function of proteins in the endocytic pathway rather than ClC-2 directly. Using a combination of immunofluorescence and biochemical studies, we confirmed that ClC-2 is internalized via dynamin-dependent endocytosis and that the change in surface expression evoked by ATP depletion is partially mimicked by inhibition of dynamin function using a dynamin dominant-negative mutant (DynK44A). Furthermore, trafficking via the early endosomal compartment occurs in part through rab5-associated vesicles and recycling of ClC-2 to the cell surface occurs through a rab11 dependent pathway. In summary, we have determined that the internalization of ClC-2 by endocytosis is inhibited by metabolic stress, highlighting the importance for understanding the molecular mechanisms mediating the endosomal trafficking of this channel.

ACRP30 is secreted from 3T3-L1 adipocytes via a Rab11-dependent pathway

Adipocytes are now known to secrete a range of adipokines that exhibit distinct biological functions. Here, we sought to understand the secretory pathways utilised by ACRP30 to the surface of adipocytes. We find that ACRP30 overlaps with adipsin in intracellular compartments distinct from Glut4, but nonetheless exhibits insulin-stimulated secretion from cells. Both adipsin and ACRP30 overlap with transferrin receptor-positive membranes, implying that the pathway of secretion involves the transferrin receptor-positive endosomal system. Consistent with this, we show that ablation of endosomes significantly inhibited the secretion of ACRP30, as did treatment of cells with Brefeldin A. In order to further probe the role of recycling endosomes on the secretion of ACRP30, we over-expressed a mutant form of Rab11, Rab11-S25N, in 3T3-L1 adipocytes and found that expression of this mutant significantly reduced basal and insulin-stimulated secretion. We also demonstrate that Arf6 also plays a role in the secretion of ACRP30. Collectively, these data implicate both Arf6 and Rab11 as crucial mediators of constitutive and insulin-stimulated secretion of ACRP30 and further suggest that recycling endosomes may play a central role in this process.