Summary of Rab35 in diseases.
Rab35 mediates membrane trafficking between the plasma membrane and the early endosomes at the cell surface. Our understanding of the cellular function of Rab35 reveals its role in development and diseases. In the developmental context, Rab35 has been shown to play an important role in regulating epithelial polarity, lumen opening, myoblast fusion, intercalation of epithelium, myelination, neurite outgrowth, and oocyte meiotic maturation. Disruption of recycling endosome mediated by Rab35 has been linked to several neurological diseases, including Parkinson’s disease and Down syndrome. In addition, because Rab35 modulates cell migration through its interaction with various effectors, Rab35 plays an important role in cancers. Lastly, the Rab35-mediated recycling endosomal pathway and exocytosis is utilized by pathogens or hijacked by pathogens to promote their infection and survival. This review summarizes the function of Rab35 in endocytosis and focuses on the role of Rab35 in the context of development and diseases.
- small G proteins
- cell migration
- protein trafficking
Rab proteins constitute the largest subset of Ras-family small guanosine triphosphates (GTPases). Over 60 mammalian Rab proteins have been identified . Rab35 is an evolutionarily conserved unique Rab GTPase that mediates membrane trafficking between the plasma membrane and endosomes in eukaryotic cells . Similar to all G proteins, Rab35 undergoes molecular switching from active GTP-bound state to inactive GDP-bound state. The activity of Rab35 is highly regulated with four different Rab35 guanine-nucleotide exchange factor (GEF) and five Rab35 GTPase-activating proteins (GAPs). Depending on which effector proteins are associated with Rab35-GTP, it can mediate many cellular functions, such as cytokinesis, phagocytosis, cell migration, and exosome release [2, 3, 4, 5, 6, 7]. Most of its cellular functions involve the regulation of Rab35 in actin polymerization, Arf6 inactivation, or phosphoinositides (PtdIns(4,5)P2) [2, 8, 9, 10]. This review briefly summarizes the regulation of Rab35 in endocytosis and focuses on Rab35 in the context of development and diseases.
2. Rab35 mediates protein trafficking
Small G proteins mediate endosomal trafficking and maintain cell surface homeostasis. Two major types of endocytosis are the clathrin-mediated endocytosis (CME) and clathrin-independent endocytosis (CIE) . CME involves the selective uptake of plasma membrane that is dependent on dynamin for vesicle scission. CIE is dynamin-independent and depends on free cholesterol at the plasma membrane; CIE does not require specific endocytic sorting sequence and is known as the bulk endocytic process . The homeostasis at the cell surface requires balanced CME and CIE and the coordinated regulation by Rab35 and Arf6 . Rab35 and Arf6 work antagonistically at the plasma membrane where Arf6 recruits the Rab35 GAP to inactivate Rab35, and Rab35 recruits the Arf6 GAP to inactivate Arf6. Thus, Rab35 from CME and the Arf6 from CIE work together to balance the two branches of the endocytic pathway .
Following endocytosis, endocytic vesicles converge on early endosomes where the cargos are sorted to be recycled or transported to late endosomes before eventually fusing with lysosomes. Activated Rab35 recruits effectors that mediate the formation of recycling tubules or vesicles. A diverse array of cargoes have been reported to undergo Rab35-dependent endocytic recycling back to the plasma membrane . Overall, Rab35 plays a conserved role in mediating endocytosis recycling after cargo internalization.
The function of Rab35 is dependent on the effector proteins that bind to the active Rab35-GTP. Several key effectors of Rab35 have been identified that reveal its cellular functions. The level of the PtdIns(4,5)P2 lipid on endosomes is mediated by Rab35, since it binds to PtdIns(4,5)P2 phosphatase, OCRL . Depletion of either Rab35 or OCRL leads to accumulation of PtdIns(4,5)P2 and F-actin binding proteins in enlarged peripheral endosome. Thus, Rab35 functions with OCRL to hydrolyze PIdIns(4,5)P2 on new endosomes and help define the lipid identity of early endosomes.
Fascin is another Rab35 effector protein. It crosslinks actin and assembles F-actin filaments into parallel bundles. Rab35-GTP recruits fascin in regulating
Rab35 and Arf6 have been found to function antagonistically in regulating membrane trafficking . Two Rab35 effectors are MICAL-L1 and ACAP2 (Arf6 GAP) that are involved in neurite outgrowth (Section 3.9.3) . Another effector protein that interacts with Rab35-GTP in regulating neurite outgrowth is RUSC2. The overexpression of the RUN domain of RUSC2 inhibits Rab35-iduced neurite outgrowth in PC12 cells . The role of RUSC2 downstream of Rab35 is not known. It is likely that the Rab35 interactome is far from complete.
3. Rab35 in development
The function of Rab35 in development has been examined mainly in the fly and worm. Various cell type such as myoblasts, fly S2 cells, oocytes, osteoclasts, neurons and oligodendrocytes have been used to study specific developmental processes. This section summarizes our current understanding of the role of Rab35 in the context of development.
3.1. Rab35 polarizes the fly tracheal seamless tubes
3.2. Rab35 regulates epithelial organ lumen opening formation
Epithelial organs such as the lungs and kidneys are composed of a polarized cell monolayer surrounding a lumen. Madin-Darby canine kidney (MDCK) cells have been used as a model to examine the establishment of epithelial polarity and lumen . It is thought that new apico-basal polarity in cysts arises from divisions of a single cell where apical transmembrane proteins are transcytosed from the plasma membrane to the cell-cell contact site. A recent study using MDCK cells indicated that Rab35 was found to directly interact with Podocalyxin (PODXL), a classical apical marker that has anti-adhesive properties that promote cell-cell repulsion at the apical membrane . Rab35 knockdown with RNAi resulted in a complete inversion of polarity so that the PODXL localizes on the membrane facing the extracellular matrix instead of the lumen . Rab35 establishes the apico-basal polarity by transporting PODXL to the site of lumen formation .
3.3. Rab35 recruits fascin to form
3.4. Rab35 functions in
During development, cell assemblies that involve coordination of cellular adhesion and shape changes are needed to form tissues and organs. Internal organs such as the palate cochlea, gut and the kidney all require tissue elongation to shape an elongated body axis of an developing animal . Cellular reshaping during organ formation requires the function of apical and junctional cytoskeletal and adhesion proteins. During
3.5. Rab35 regulates mouse oocyte meiosis
During meiosis, the oocytes undergo nuclear and cytoplasmic maturation where the migration of intracellular components such as spindle, mitochondria, and cortical granules is important for subsequent embryonic development. Rab35 localizes in the ooplasm at the germinal vesicle (GV) stage . After germinal vesicle breakdown (GVBD), Rab35 is distributed at the spindle and colocalizes with α-tubulin. Rab35 RNAi treated oocytes displayed abnormal spindle morphology with multiple poles of spindle components, indicating that Rab35 regulates mouse oocyte spindle formation . In addition, Rab35 RNAi and antibody blocking experiments indicated that GVBD is not affected, but polar body extrusion defect was observed. Overall, Rab35 was found to be important for forming spindles of oocytes during oocyte meiotic maturation and activation .
3.6. Terminal steps of cytokinesis is regulated by Rab35
Intracellular transport is essential for animal cytokinesis, with both the secretory and the endocytic pathways being implicated in the late phase of cytokinesis. Previously Rabs important in cytokinesis was screened with RNAi in S2 cells, in search for binucleated cells that failed to undergo cytokinesis . Rab35 is found to be required for the stabilization of the cytokinesis bridge connecting the daughter cells after furrow ingression as well as the abscission . The proposed mechanism of Rab35 here is that Rab35 controls the localization of phosphatidylinositol 4,5-bis phosphate (PIP2) and SEPT2 at the bridge which are required for the stability of cytokinesis completion [2, 24, 25]. Rab35-mediated endocytic recycling is important for the stabilization of the late stage cytokinesis and abscission.
3.7. Rab35 regulates endocytic recycling of yolk receptor
Previously genetic mutants of endocytosis were identified in
3.8. Rab35 mediates myoblast fusion
During embryonic development, assembly and disassembly of cadherins play an important role in morphogenesis, cell differentiation, growth and migration . Rab35 regulates cadherin trafficking and stabilization at cell-to-cell contacts to mediate myoblast fusion . Rab35-S22N DN and RNAi results indicated a reduction of N- and M-cadherin at cell-to-cell contacts and increased accumulation in intracellular vacuoles. Rab35 RNAi and DN inhibited myoblast differentiation by preventing myoblast fusion to form myoblasts . Overexpression of Rab35-WT and constitutively active Rab35-Q67L indicated their colocalization at the plasma membrane with PI(4,5)P2, but no perturbation of PI(4,5)P2 was observed . The proposed mechanism is that Rab35 function is required for PI(4,5)P2 production which stabilizes cadherin at cell-cell contact sites. Taken together, these results indicate that Rab35 regulates cadherin-dependent adherens junction formation and myoblast fusion .
3.9. Rab35 in the nervous system
3.9.1. Rab35 suppresses oligodendrocyte differentiation
During development of the central nervous system, oligodendrocytes precursor cells undergo cell division and migrate along axons where oligodendrocytes differentiate to wrap axons with myelin sheaths . The dynamic morphological changes are in part mediated by small GTPase signaling. The regulatory role of Rab35 in oligodendrocyte differentiation was examined in FBD-102b (mouse oligodendroglial cells) cells . Rab35 activates its effector protein ACAP2 (a Arf6-GAP) to deactivate Arf6, which inhibits FBD-102b differentiation . Consistent with this result, knockdown of Arf6 with RNAi inhibits oligodendrocyte differentiation. In oligodendrocyte and neuronal cocultures, knockdown of Rab35 or ACAP2 promotes myelination, and inhibition of cytohesin-2 (Arf6-GAP) or Arf6 knockdown inhibits myelination . These studies revealed that Rab35 and Arf6 function antagonistically in regulating the differentiation of oligodendrocyte and myelination.
3.9.2. Rab35 coordinates synaptic vesicle trafficking and turnover
At least 30 out of the 60 mammalian Rab GTPases are associated with synaptic vesicle (SV) pools . Antagonistic and synergistic functions of molecules within the Rab35 and Arf6 signaling network are necessary for regulating SV protein trafficking, degradation, and neurotransmitter release. Depending on neuronal activity, SVs may either get exocytosed when Arf6 is activated, or SVs can get recruited to presynaptic endosomes when Rab35 is activated . Dysfunction of this signaling network may induce neurologic and neurodegenerative diseases. The molecular mechanism of SV protein turnover was further defined by using the rat hippocampal neurons . Rab35 degrades SV proteins via the endosomal sorting complex required for transport (ESCRT) pathway, by recruiting Rab35 effector and ESCRT protein, Hrs. Upon neuronal stimulation, ESCRT proteins are recruited to SV pools to degrade specific SV proteins .
In addition, Rab35 and its GAP, Skywalker (Sky), were found to be key players in the endosomal sorting/recycling of SV proteins . Sky was identified to facilitate endosomal trafficking of synaptic vesicles at
3.9.3. Rab35 mediates neurite outgrowth
Rab35 has been shown to promote neurite outgrowth of PC12 cells in response to nerve growth factor (NGF) stimulation [35, 36]. Upon nerve growth factor (NGF) stimulation, Rab35 accumulates in Arf6-positive endosomes . Both Rab35 and Arf6 work antagonistically to regulate neurite outgrowth. The same Rab35 effector, ACAP2 (or centaurin-β2) that regulates the differentiation of oligodendrocytes (Section 3.9.1), is recruited to the Arf6-positive endosomes in a Rab-35-dependent manner upon NGF stimulation. The Arf6-GAP activity of ACAP2, leading to Arf6 inactivation, was required for NGF-induced neurite outgrowth . In addition, Rab35 was found to form a tripartite structure with MICAL-L1 and ACAP2 and recruit them to Arf6-positive endosomes in response to NGF. MICAL-L1 and ACAP2 cooperatively recruit EHD1, which belongs to the dynamin-like C-terminal Eps15 homology domain protein family . EHD1 promotes membrane trafficking of various receptors, mainly from recycling endosomes to the plasma membrane. EHD1 functions as molecular scissors that facilitate fission of vesicles from recycling endosomes via its ATPase activity. Knockdown of Rab35, MICAL-L1, ACAP2, and EHD1 all resulted in shortened neurite outgrowth, indicating the importance of each of these components [36, 38]. In summary, Rab35 recruits and coordinates MICAL-L1 and ACAP2 to Arf6-positive endosomes. At the same time, EHD1 is recruited by binding to MICAL-L1 where it may facilitate neurite tip outward growth by mediating fission of vesicles that target to neurite tips from recycling endosomes during neurite outgrowth.
Rab35 has been proposed to act as a master Rab that determines the intracellular localization of MICAL-L1, which functions as a scaffold for other recruited Rabs . Upon NGF stimulation, Rab35 localizes to Arf6-positive recycling endosomes and recruits MICAL-L1, which interacts with Rabs 8, 13, and 36 . Each of these recruited Rabs functions in a non-redundant manner downstream of Rab35 and MICAL-L1 in regulating neurite outgrowth . Knockdown of individual MICAL-L1 interacting Rabs did not alter MICAL-L1 localization but inhibited NGF-induced neurite outgrowth. Overall, the NGF stimulation activates Rab35 which recruits several other Rabs at recycling endosomes that supply membranes and proteins to enable neurite outgrowth.
3.9.4. Rab35 functions in axon elongation
Neurons acquire an asymmetric morphology during embryonic development to establish neuronal polarization, where a single axon and several dendrites are formed . Neuronal polarized trafficking is dependent on the supply of membrane needed to cell expansion and the differential distribution of proteins. This process involves Rab35 and its regulators. Rab35 was found to function in axon elongation that is regulated by p53-related protein kinase, or PRPK . PRPK is a negative regulator of Rab35 that promotes the degradation of Rab35 via the ubiquitin proteasome degradation pathway. Another protein, microtubule-associated protein 1B (MAP1B), interacts with PRPK to inhibit its degradation of Rab35 . MAP1B is necessary for proper axon outgrowth, as decreased MAP1B expression reduces axon length. MAP1B knock out is rescued by Rab35 overexpression or PRPK inactivation. Neurons overexpressing Rab35 WT and active Rab35-Q67L exhibited a significant increase in exon length. In contrast, Rab35-S22N DN transfected neurons had reduced axon length. In addition, Rab35 activates Cdc42 by either direct activation of Cdc42 or transporting vesicles containing polarity determinants to the elongating exons . Overall, these results indicate that Rab35 is critical for mediating neuronal polarization trafficking to elongate axons.
4. Rab35 in diseases
Disruption of recycling endosome mediated by Rab35 has been linked to several neurological diseases, including Parkinson’s disease and Down syndrome [41, 42]. In addition, because Rab35 modulates cell migration through its interaction with Wnt/Dvl signaling pathway and F-actin modulators, Rab35 plays an important role in cancers (see Section 4.3). Lastly, the Rab35-mediated recycling endosomal and exocytosis pathways are used by pathogens to promote their infections and survival (see Section 4.4). This section summarizes the role of Rab35 in various diseases (Table 1).
|Disease||Function||Potential molecular mechanism||Ref|
|Parkinson’s disease||Endocyclic recycling of α-synuclein||Unknown|||
|Down syndrome||Exosome release||Unknown|||
|Breast cancer||Promotes cell migration||Activation of Rac1 via Wnt5a/Dvl2.|
Active Rab35 and MICAL1 generate ROS and activate Akt pathway
|Lung cancer||Enhance cell polarization and migration||Rab35 mediates interaction of RUSC2 and GIT2; mediate GIT2 phosphorylation|||
|Allergy-induced asthma||Delayed TCR recycling; increase cytokines||DENND1B interacts with AP-2 to mediate Rab35 GTP exchange|||
|Amoebic colitis ||Uses Rab35 to phagocytose RBCs||Unknown|||
|Uropathogenic ||Iron acquisition; lysosome evasion||Exploits host TfR1 to acquire iron|||
|Enterohemorrhagic ||Inhibit host endocyclic recycling pathway||Bacteria EspG interacts with Arf6-GTP|||
|Legionnaires disease||Evade fusion with host lysosomes||LepB stimulates GTP hydrolysis on Rab35|
AnkX modifies Rab35 with phosphocholine
|Antrax ||Endocytosis and release anthrax lethal toxin||Rab35 mediates MAPKK cleavage and exosome formation|||
4.1. Rab35 may be involved in Parkinson’s disease
Parkinson’s disease (PD) is a neurodegenerative disease in which the patient’s dopaminergic neurons in the substantia nigra are impaired . Lewy bodies composed of abnormal α-synuclein accumulate in substantia nigra neurons of PD patients . The serum levels of Rab35 was high in PD patients and in the substantia nigra of mice models for PD . Overexpression of Rab35 in SH-SY5Y cells (cell line model to study neuronal function) resulted in increased aggregation and secretion of α-synuclein . Although no detailed mechanism of Rab35 in the pathogenesis of PD is known, Rab35 may participate in the processing and endocyclic recycling of α-synuclein . This proposed role of Rab35 in PD is in part supported by several studies that have shown α-synuclein to interact with other Rab proteins [45, 46]. The level of Rab35 in patient serum may be useful in the diagnoses of different Parkinsonian disorders.
In addition, activation of leucine-rich repeat protein kinase 2 (LRRK2), caused by autosomal dominant missense mutation, predisposes patients to PD . LRRK2 is a Rab GTPase that has been found to phosphorylate 14 Rab proteins, including Rab35 . Specific LRRK2 antibodies that recognize phosphorylated forms of Rab proteins have been developed to examine how LRRK2 and Rab proteins contribute to PD and may serve as a potential therapeutic tool [49, 50].
4.2. Rab35 controls exosome secretion in Down syndrome patients
Early endosomal abnormalities have been correlated to developmental brain defects in Alzheimer’s disease (AD) and Down syndrome patients (DS) . In the neurons of these patients, their endosomes are aberrantly numerous and enlarged with accumulated materials that lead to neuronal vulnerability and degeneration . Early endosomes are the first vesicular compartment along the endocytic pathway where internalized cargos are delivered to late endosomes or multivesicular bodies (MVBs) for sorting to either lysosomes for degradation or to the extracellular space via exosomes release (EVs). The docking of MVBs to the plasma membrane is regulated by Rab35 . In human brain homogenates, Rab35 proteins were at a higher level in DS patients compared to controls using western blotting detection . In addition, DS patients and Ts2 mice (murine model for DS) have higher levels of exosome-enriched EVs and Rab35 in the brain extracellular space . Rab35 is proposed to play a protective role in mediating exosome release to relieve neurons of the toxic materials in neuronal endosomes in DS and AD patients .
4.3. Rab35 functions in cell migration and cancers
Rab35 has been shown to interact with effector proteins that are involved in cell adhesion and cell migration which are key processes that are disrupted in cancer. Rab35 is required for Wnt5a/Dvl2-induced Rac1 activation and cell migration in MCF-7 breast cancer cells . Upon
In another study, active Rab35 and its effector protein, MICAL1, control cell invasive phenotype in breast cancer cells . MICAL1 has been shown to upregulate reactive oxygen species (ROS) in HeLa cells and phosphorylate proteins leading to malignancies and metastasis. Breast cancer cells receive signals from their microenvironment, such as epidermal growth factor (EGF), LPA and hypoxia, ROS level in cells may increase and functions as second messengers in intracellular signaling cascades to induce their metastasis . Upon stimulation of EGF, Rab35 levels increased in MCF-7 cells . Rab35 knockdown using siRNA in MCF-7 cells showed a dramatic decrease in cell invasion, demonstrating that Rab35 was required for EGF-induced invasion in breast cancer cells . Transfection of cells with siRab35 or siMICAL1 led to decreased ROS, indicating that both Rab35 and MICAL1 are required for ROS generation. Further, the generated ROS was found to activate the PI3K/Akt pathway which also plays a key role in migratory potential regulation. Consistent with this result, knockdown of Rab35 or MICAL1 by RNAi resulted in decreased phosphorylated Akt (P-Akt) . Similarly, P-Akt was higher when MICAL1 or Rab35-GTP (active) were overexpressed in MCF-7 cells. Together, these results revealed that Rab35 and MICAL1 promote ROS production which leads to PI3K/Akt signaling activation, resulting in increased breast cancer cell migration and invasion .
Consistent with the role of Rab35 in activating the Akt signaling pathway, an earlier study identified that Rab35 functions downstream of growth factor receptors and upstream of the Akt signaling pathway . Using lentiviruses that express short hairpin RNAs (shRNAs) targeting genes coding for all known G-proteins and lipid/protein, Rab35 knockdown was found to downregulate Akt phosphorylation . Furthermore, the PI3K-dependent phosphorylation of FOXO1/3A was also decreased in cells depleted of Rab35. Wild type, constitutively active Rab35-Q67L and DN Rab35-S22N was each expressed in cell lines and only the Rab35-Q67L active form bound to and activate FOX01/3A and the P13K/AKT signaling pathway . Based on missense mutations previously identified in the proto-oncogene KRAS in myeloid leukemia patients and colorectal tumors, Rab35 with A151T and F161L mutations were tested for their effect on AKT signaling [56, 57]. Interestingly, Rab35-A151T and Rab35-F161L mutants expressed stably in NIH-3T3 cells also resulted in elevated AKT phosphorylation levels, indicating that these gain-of-function alleles are sufficient to activate PI3K/AKT signaling . Therefore, these studies demonstrated that Rab35 activates AKT signaling in cancer cells to suppress apoptosis and aid in cell transformation.
Rab35 plays an important role in non-small cell lung cancer (NSCLC) cell migration by regulating the interaction of RUSC2 (Rab35 effector protein) and GIT2 (Arf6-GAP) . Both RUSC2 and GITS2 have been found to regulate cell polarity and directional cell migration [16, 59]. The function of RUSC2 is not well characterized and may participate in vesicle-mediated transport and secretory pathway to regulate directional migration . GIT2 interacts with paxillin to mediate normal cell spreading and lamellipodia formation . Upon EGF stimulation, Rab35 is activated and promotes the binding of RUSC2 to the non-phosphorylated form of GIT2 . Knockdown of Rab35 or RUSC2 by RNAi resulted in decreased GIT2 phosphorylation and its half-life, indicating that Rab35 and RUSC2 are each essential for GIT2 phosphorylation and stability . The phosphorylated form of GIT2 is released from RUSC2 and localizes to the plasma membrane to mediate cell migration . Collectively, these data indicate that upon EGF stimulation, active Rab35 promotes the interaction of RUSC2 and GIT2, the intracellular stabilization and phosphorylation of GIT2, and lung cancer cell polarization and cell migration .
4.4. Rab35 may regulate T-cell receptor signaling
Upon receptor complex activation, the duration of signaling is affected by alterations in receptor internalization, recycling, and degradation . The prolonged activation of T-cell receptor (TCR) on immune TH2 cells promotes allergic asthma . TCRs within the plasma membrane of TH2 cells are dynamically regulated through endocytosis and recycling [64, 65]. The role of Rab35-GEF, DENND1B, in allergic asthma was investigated in mice . The independent knockdown of DENN1B, Rab35, or clathrin adaptor AP-2 resulted in delayed TCR downmodulation after its activation . This in turn resulted in aberrant, prolonged TCR signaling and increased cytokine secretion of IL-4, IL-5, and IL-13 in TH2 cells. The ability of DENND1B to interact with AP-2 and mediate Rab35 GTP exchange is required for optimal regulation of surface TCR signaling in TH2 cells . These findings were consistent with enhanced
4.5. Pathogens use the Rab35 pathway during infections
Rab35 has been demonstrated to be involved in the process of erythrophagocytosis of
An additional function of the Rab35 recruitment from the host is to promote UPEC survival by preventing the fusion of UPEC-containing vesicles with the hosts’ degradative lysosomes . UPECs colocalized with lysosomes in normal and Rab35-deleted cells in late endosomes and lysosomes. In Rab35 knockdown BECs, a significantly higher number of intracellular UPECs colocalized with the lysosomal marker where UPECs are destroyed. Thus, UPECs utilize the host Rab35 mediated vesicular trafficking pathways to enhance its iron acquisition and prevent lysosomal degradation within the bladder epithelial cells during infection .
Rab35 may play a role in the pathogenesis of
Rab35 is a highly conserved small GTPase that is the only Rab that mediates endosomal recycling of target proteins between the plasma membrane and the early endosomes. Further understanding of the interactome of Rab35 will elucidate additional functions of Rab35 in the context of development and disease.
Conflict of interest
The authors declare no conflict of interest.