an-enhanced-green-fluorescence-protein-based-assay-for-studying

An Enhanced Green Fluorescence Protein-based Assay for Studying Neurite Outgrowth in Primary Neurons

Neurite outgrowth is a fundamental event in the formation of the neural circuits during nervous system development. Severe neurite damage and synaptic ...

density-gradient-ultracentrifugation-for-investigating-endocytic

Density Gradient Ultracentrifugation for Investigating Endocytic Recycling in Mammalian Cells

Endosomal trafficking is an essential cellular process that regulates a broad range of biological events. Proteins are internalized from the plasma ...

The Chinese University of Hong Kong, Hong Kong Special Administrative Region

The Chinese University of Hong Kong

Ras-related C3 botulinum toxin substrate 1 (Rac1) is a member of the Rho family of GTPases that functions as a molecular switch to regulate many important cellular events including actin cytoskeleton remodeling during neurite outgrowth. Engulfment and cell motility 1 (ELMO1)-dedicator of cytokinesis 1 (DOCK180) is a bipartite guanine nucleotide exchange factor (GEF) complex that has been reported to activate Rac1 on the plasma membrane (PM). Emerging evidence suggests that the small GTPase ADP ribosylation factor 6 (ARF6) activates Rac1 via the ELMO1/DOCK180 complex. However, the exact mechanism by which ARF6 triggers ELMO1/DOCK180-mediated Rac1 signaling remains unclear. Here, we report that the neuronal scaffold protein FE65 serves as a functional link between ARF6 and ELMO1, allowing the formation of a multimeric signaling complex. Interfering with formation of this complex by transfecting either FE65-binding-defective mutants or FE65 siRNA attenuates both ARF6-ELMO1-mediated Rac1 activation and neurite elongation. Notably, the PM trafficking of ELMO1 is markedly decreased in cells with suppressed expression of either FE65 or ARF6. Likewise, this process is attenuated in the FE65-binding-defective mutants transfected cells. Moreover, overexpression of FE65 increases the amount of ELMO1 in the recycling endosome, an organelle responsible for returning proteins to the PM, whereas knockout of FE65 shows opposite effect. Together, our data indicates that FE65 potentiates ARF6-Rac1 signaling by orchestrating ARF6 and ELMO1 to promote the PM trafficking of ELMO1 via the endosomal recycling pathway, and thus, promotes Rac1-mediated neurite outgrowth.

ARF6-Rac1 signaling-mediated neurite outgrowth is potentiated by the neuronal adaptor FE65 through orchestrating ARF6 and ELMO1.

Intramolecular interaction is a common mechanism that regulates protein activities. Conventionally, such interactions are investigated by classical  biochemical assays. Here, we describe a protocol for studying the intramolecular interaction of cell motility and engulfment 1 (ELMO1) in mammalian cells by using proximity ligation assay (PLA). PLA is a specific and sensitive method that allows the observation of interacting proteins by target-specific antibody detection coupled to rolling circle amplification. ELMO1 is the regulatory subunit of ELMO1-dedicator of cytokinesis 180 (DOCK180) bipartite Rac1 guanine nucleotide exchange factor (GEF) which adopts a closed autoinhibitory conformation via an intramolecular interaction of its N-terminal ELMO inhibitory domain (EID) and C-terminal ELMO autoregulatory domain (EAD). In the assay, PLA signals are detected in cells transfected with ELMO1 and ELMO1 fragments. Moreover, overexpression of FE65, a neuronal adaptor which has been shown to disrupt ELMO1 intramolecular interaction, reduces the PLA signals of the two ELMO1 fragments significantly. Together, our results demonstrate that PLA can be employed for studying protein intramolecular interaction.

Wai Wa Ray Chan

School of Life Sciences

The Chinese University of Hong Kong, Hong Kong Special Administrative Region

An Enhanced Green Fluorescence Protein-based Assay for Studying Neurite Outgrowth in Primary Neurons

Density Gradient Ultracentrifugation for Investigating Endocytic Recycling in Mammalian Cells

Wai Wa Ray Chan

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}School of Life Sciences

The Chinese University of Hong Kong, Hong Kong Special Administrative Region

An Enhanced Green Fluorescence Protein-based Assay for Studying Neurite Outgrowth in Primary Neurons

Density Gradient Ultracentrifugation for Investigating Endocytic Recycling in Mammalian Cells

School of Life Sciences