RAB35 Human

What is RAB35 and what are its primary cellular functions?

RAB35 is a member of the Rab family of small GTPases that functions as a master regulator of membrane trafficking. It cycles between inactive GDP-bound and active GTP-bound states, regulated by specific guanine-nucleotide exchange factors (GEFs) and GTPase-activating proteins (GAPs) . Functionally, RAB35 plays critical roles in:

• Endocytic recycling of cargo at the plasma membrane-endosome interface

• Regulation of actin cytoskeleton dynamics and organization

• Formation of circular dorsal ruffles (CDRs) during cell migration

• Control of epithelial cell adhesion and polarity

• Cytokinesis during cell division

• Regulation of primary cilia length and composition

The multifaceted role of RAB35 establishes it as a critical node in cellular homeostasis, with its dysfunction potentially contributing to developmental abnormalities and disease states.

How is RAB35 activity regulated in human cells?

RAB35 activity is primarily regulated through a cycle of GTP binding (activation) and hydrolysis (inactivation):

• Activation: Mediated by GEFs from the connecdenn/DENND1 family, which catalyze the exchange of GDP for GTP

• Inactivation: Facilitated by GAPs, particularly those from the TBC1D10/EPI64 family, which promote GTP hydrolysis

• Membrane association: RAB35 associates with membranes via prenylation of its C-terminus

• Effector binding: In its GTP-bound state, RAB35 interacts with numerous effector proteins that mediate its downstream functions

This regulatory cycle enables precise temporal and spatial control of RAB35-dependent processes, allowing for dynamic modulation of membrane trafficking and cytoskeletal organization in response to cellular needs.

What phenotypes result from RAB35 loss in mammalian models?

Studies using knockout mouse models have revealed that RAB35 is essential for mammalian development and tissue homeostasis:

• Embryonic lethality: Congenital RAB35 knockout mice exhibit embryonic arrest at E7.5 with cardiac edema, demonstrating its essential role in early development

• Kidney and ureter defects: Conditional deletion of RAB35 during gestation or postnatally causes non-obstructive hydronephrosis associated with:

  • Disrupted actin cytoskeletal architecture

  • Altered Arf6 epithelial polarity

  • Reduced adherens junctions

  • Loss of tight junction formation

  • Defects in EGFR expression and localization

  • Shortened primary cilia

• Cellular migration defects: Loss of RAB35 impairs directed chemotactic migration and chemoinvasion in response to growth factor gradients

These phenotypes highlight RAB35's critical importance in developmental processes and tissue maintenance, particularly in epithelial structures.

How does RAB35 coordinate with the PI3K pathway to regulate chemotaxis?

RAB35 functions as a molecular determinant for controlling an excitable, oscillatory system that directs growth factor-mediated chemotaxis. The molecular coordination involves:

• Direct regulation: RAB35 directly regulates the activity of the p85/PI3K polarity axis

• Wave formation: RAB35 is necessary for the formation of growth factor-induced waves of circular dorsal ruffles (CDRs)

• Excitable system properties: When activated, RAB35 induces recurrent and polarized CDRs that travel as propagating waves, exhibiting properties of an excitable system

• Cell steering mechanism: This oscillatory behavior can be biased to control directional cell movement, effectively functioning as a "steering wheel" for chemotaxis

This coordination between RAB35 and PI3K provides cells with the ability to interpret noisy, shallow gradients of soluble cues, which is essential for effective chemotactic navigation.

What is the relationship between RAB35 and Arf6 in regulating epithelial cell polarity?

RAB35 and Arf6 exhibit an antagonistic relationship in regulating epithelial cell polarity and membrane protein trafficking:

• Antagonistic regulation: RAB35 antagonizes Arf6, which functions to internalize surface proteins such as EGFR that inhibit cell adhesion and promote cell migration

• Polarity maintenance: In RAB35 mutant kidney cells, Arf6 localization is disrupted, leading to altered epithelial polarity

• Boundary formation: RAB35 appears to create an Arf6 apical boundary through regulation of Arf6 activity, which influences actin organization and E-cadherin localization on the baso-lateral membrane

• Junction integrity: This regulation is necessary to maintain epithelial adherens junctions and subsequently tight junctions, as well as the localization of membrane receptors like EGFR

This antagonistic relationship provides a mechanism for the precise control of epithelial cell polarity, which is essential for tissue architecture and function.

How do the different RAB35 effector proteins mediate its diverse cellular functions?

RAB35 interacts with a plethora of effector proteins to mediate its diverse cellular functions:

• Actin regulation effectors: Interact with RAB35 to control actin organization, critical for processes like cell migration, adhesion, and cytokinesis

• Membrane trafficking effectors: Mediate RAB35's role in cargo recycling at endosomes

• Cytoskeletal effectors: Enable RAB35 to influence cell shape, movement, and division

• Polarity signaling effectors: Connect RAB35 to signaling pathways that establish and maintain cell polarity

Each effector protein likely mediates a specific subset of RAB35 functions, allowing this single GTPase to orchestrate diverse cellular processes in a coordinated manner. The specificity of these interactions and their regulation remains an active area of research.

What is the role of RAB35 in embryonic development and tissue homeostasis?

RAB35 plays essential roles in both embryonic development and the maintenance of tissue homeostasis:

• Early embryogenesis: Congenital loss of RAB35 in mice causes embryonic arrest at E7.5, suggesting critical functions in gastrulation and early organogenesis

• Epithelial integrity: RAB35 maintains epithelial structures in the urogenital system by regulating:

  • E-cadherin expression and localization

  • Formation and maintenance of cell-cell junctions

  • Actin cytoskeletal architecture

  • Proper localization of polarity markers like Arf6

• Organ function: In the kidney and ureter, RAB35 is essential for maintaining normal architecture and function, with its loss leading to hydronephrosis

• Cell differentiation: RAB35 appears to influence cell differentiation, as its loss leads to disrupted cell differentiation in kidney and ureter tissues

These roles highlight RAB35 as a critical regulator of both developmental processes and tissue maintenance throughout life.

How does RAB35 dysfunction contribute to disease pathogenesis?

While the search results don't provide direct information on human diseases associated with RAB35 dysfunction, the mouse model findings suggest several potential pathological mechanisms:

• Developmental disorders: Given its essential role in embryonic development, RAB35 mutations could contribute to congenital abnormalities

• Kidney disease: The non-obstructive hydronephrosis observed in RAB35 mutant mice suggests RAB35 dysfunction may contribute to similar kidney pathologies in humans

• Cancer progression: RAB35 provides a novel link between Rab and Arf family GTPases with implications for tumor formation and invasiveness

• Cell migration disorders: The role of RAB35 in chemotaxis suggests its dysfunction could affect processes requiring directed cell migration, such as wound healing or immune responses

These potential disease associations make RAB35 an important target for research in developmental biology, nephrology, and oncology.

What is the tissue-specific expression pattern of RAB35 and how does it relate to function?

RAB35 exhibits a dynamic expression pattern that changes during development and varies across tissues:

• Embryonic expression: At E8.5, RAB35 is ubiquitously expressed, consistent with its essential role in early development

• Postnatal expression: By P7, RAB35 expression becomes more restricted:

  • Highest expression in kidney, ureter, and testes

  • Modest expression in lung and liver

  • Minimal expression in heart and pancreas

• Cellular distribution: Within expressing tissues, RAB35 is enriched in:

  • Epithelial cells

  • Ciliated cells (e.g., biliary epithelial cells and spermatozoa)

• Intra-organ variation: In the kidney, RAB35 expression is not uniform, with greater activity in the cortex and renal papilla than in the medulla

This expression pattern aligns with the observed phenotypes in RAB35 mutant mice, particularly the prominent kidney and ureter defects, and suggests tissue-specific functions that may be explored in future research.

What are the optimal approaches for studying RAB35 activation and inactivation dynamics in living cells?

Investigating RAB35 activation and inactivation dynamics requires specialized techniques:

• FRET-based biosensors: Design and implementation of fluorescence resonance energy transfer biosensors can allow real-time visualization of RAB35 activation states in living cells

• Optogenetic tools: Light-controlled activation or inactivation of RAB35 enables precise temporal and spatial manipulation of its activity

• GTP-locked and GDP-locked mutants: Generation of constitutively active (Q67L) or inactive (S22N) RAB35 mutants for functional studies

• GEF/GAP manipulation: Overexpression or knockdown of specific RAB35 GEFs (connecdenn/DENND1 family) or GAPs (TBC1D10/EPI64 family) to modulate activation dynamics

• Live cell imaging: Combined with fluorescently tagged RAB35 and its regulators/effectors to track subcellular localization and dynamics

These approaches can provide complementary insights into the spatial and temporal aspects of RAB35 regulation in different cellular contexts.

What experimental strategies can resolve contradictory findings regarding RAB35 function?

Resolving contradictory findings in RAB35 research requires systematic experimental approaches:

• Context-dependent analysis: Investigate how cell type, developmental stage, or experimental conditions might influence RAB35 function

• Acute vs. chronic manipulation: Compare acute depletion (e.g., using degron-based approaches) with long-term genetic knockout to distinguish between direct effects and compensatory responses

• Isoform-specific targeting: Ensure that experimental manipulations target specific RAB35 isoforms if multiple exist

• Effector-specific analysis: Systematically examine the contributions of different RAB35 effectors to specific cellular processes

• Combined in vitro and in vivo approaches: Validate findings across multiple experimental systems, from cell lines to organoids to animal models

• Quantitative measurements: Develop quantitative assays for specific RAB35 functions to allow more precise comparisons across studies

This systematic approach can help reconcile seemingly contradictory findings by uncovering the specific conditions under which different RAB35 functions predominate.

How can researchers effectively investigate the crosstalk between RAB35 and other GTPases?

Investigating crosstalk between RAB35 and other GTPases such as Arf6 requires specialized experimental strategies:

• Sequential or simultaneous manipulation: Knockdown or overexpression of multiple GTPases in defined sequences to determine epistatic relationships

• Proximity labeling approaches: BioID or APEX2-based proximity labeling to identify proteins in the vicinity of active RAB35

• Co-immunoprecipitation studies: Investigate physical interactions between RAB35, its regulators, and other GTPases

• Activity assays: Measure the activity of one GTPase while manipulating another to establish regulatory relationships

• High-resolution microscopy: Super-resolution or FRET microscopy to visualize co-localization or direct interactions between different GTPases

• Mathematical modeling: Develop computational models of GTPase networks to predict emergent behaviors and crosstalk mechanisms

These approaches can reveal how RAB35 functions within broader GTPase networks to coordinate complex cellular processes such as membrane trafficking, cytoskeletal dynamics, and cell polarity.