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Clostridium botulinum neurotoxin serotype A (BoNT/A) is a potent neurotoxin that serves as an effective therapeutic for several neuromuscular disorders via induction of temporary muscular paralysis. Specific binding and internalization of BoNT/A into neuronal cells is mediated by its binding domain (HC/A), which binds to gangliosides, including GT1b, and protein cell surface receptors, including SV2. Previously, recombinant HC/A was also shown to bind to FGFR3. As FGFR dimerization is an indirect measure of ligand-receptor binding, an FCS & TIRF receptor dimerization assay was developed to measure rHC/A-induced dimerization of fluorescently tagged FGFR subtypes (FGFR1-3) in cells. rHC/A dimerized FGFR subtypes in the rank order FGFR3c (EC50 ≈ 27 nM) > FGFR2b (EC50 ≈ 70 nM) > FGFR1c (EC50 ≈ 163 nM); rHC/A dimerized FGFR3c with similar potency as the native FGFR3c ligand, FGF9 (EC50 ≈ 18 nM). Mutating the ganglioside binding site in HC/A, or removal of GT1b from the media, resulted in decreased dimerization. Interestingly, reduced dimerization was also observed with an SV2 mutant variant of HC/A. Overall, the results suggest that the FCS & TIRF receptor dimerization assay can assess FGFR dimerization with known and novel ligands and support a model wherein HC/A, either directly or indirectly, interacts with FGFRs and induces receptor dimerization.

Mutations within the Leucine-Rich Repeat Kinase 2 (LRRK2) gene are the most common genetic cause of autosomal and sporadic Parkinson&rsquo, s disease (PD). LRRK2 is a large multidomain kinase that has reported interactions with several membrane proteins, including Rab and Endophilin, and has recently been proposed to function as a regulator of vesicular trafficking. It is unclear whether or how the spatiotemporal organization of the protein is altered due to LRRK2 activity. Therefore, we utilized fluctuation-based microscopy along with FLIM/FRET to examine the cellular properties and membrane recruitment of WT LRRK2-GFP (WT) and the PD mutant G2019S LRRK2-GFP (G2019S). We show that both variants can be separated into two distinct populations within the cytosol, a freely diffusing population associated with monomer/dimer species and a slower, likely vesicle-bound population. G2019S shows a significantly higher propensity to self-associate in both the cytosol and membrane regions when compared to WT. G2019S expression also resulted in increased hetero-interactions with Endophilin A1 (EndoA1), reduced cellular vesicles, and altered clathrin puncta dynamics associated with the plasma membrane. This finding was associated with a reduction in transferrin endocytosis in cells expressing G2019S, which indicates disruption of endocytic protein recruitment near the plasma membrane. Overall, this study uncovered multiple dynamic alterations to the LRRK2 protein as a result of the G2019S mutation&mdash, all of which could lead to neurodegeneration associated with PD.

Exosomes, vehicles for intercellular communication, are formed intracellularly within multivesicular bodies (MVBs) and are released upon fusion with the plasma membrane. For their biogenesis, proper cargo loading to exosomes and vesicle traffic for extracellular release are required. Previously we showed that the L-type lectin, LMAN2, limits trans-Golgi Network (TGN)-to-endosomes traffic of GPRC5B, an exosome cargo protein, for exosome release. Here, we identified that the protein deacetylase sirtuin 2 (SIRT2) as a novel interactor of LMAN2. Loss of SIRT2 expression resulted in exosomal release of LMAN2, a Golgi resident protein, along with increased exosomal release of GPRC5B. Furthermore, knockout of SIRT2 increased total number of extracellular vesicles (EVs), indicating increased MVB-to-EV flux. While knockout of SIRT1 increased EV release with enlarged late endolysosome, knockout of SIRT2 did not exhibit endolysosome enlargement for increased EV release. Taken together, our study suggests that SIRT2 regulates cargo loading to MVBs and MVB-to-EV flux through a mechanism distinct from that of SIRT1.

Clostridium botulinum neurotoxin serotype A (BoNT/A) is a potent neurotoxin that also serves as an effective therapeutic for a variety of neuromuscular and glandular diseases and disorders. The observed pharmacological effect of BoNT/A is due to specific targeting and entry into motor nerve terminals within muscles followed by cleavage of the SNARE protein, SNAP-25, inducing a block in neurotransmission and temporary muscular paralysis. Specific binding and internalization of BoNT/A into neuronal cells are mediated by its binding domain (HC/A), which binds to GT1b ganglioside and protein cell surface receptors. Previously, fibroblast growth factor receptor 3 (FGFR3) was identified as a BoNT/A receptor, in addition to synaptic vesicle protein (SV2). To further study BoNT/A interactions with FGFRs, an FCS & TIRF receptor dimerization assay was developed to measure dimerization of FGFRs in live cells, as FGFR dimerization can be considered an indirect measure for receptor-ligand binding interaction and downstream signaling. The ability of HC/A to facilitate dimerization of three FGFR subtypes (FGFR1-3) was assessed. Recombinant HC/A (rHC/A) was shown to dimerize FGFR subtypes in the rank order FGFR3c > FGFR2b > FGFR1c. With potencies (EC50 values) defined as the concentration of ligand required to dimerize 50% of the receptors, wild type rHC/A dimerized FGFR3c with an EC50 of 24 nM, similar to FGF9, a native FGFR3c ligand, which had an EC50 of 15 nM, while FGFR1c and FGFR2b required higher rHC/A concentrations (≥100 nM and 68 nM, respectively). Furthermore, addition of the GT1b ganglioside to the culture media resulted in increased dimerization, whereas a ganglioside mutant variant of HC/A (rHC/A W1266L;Y1267S) showed decreased dimerization. Interestingly, reduced dimerization was also observed with an SV2 mutant variant of HC/A (rHC/A T1145A;T1146A). These results support a model wherein BoNT/A interacts with FGFRs, gangliosides, and SV2 on the cell surface to facilitate cell uptake.

Activity-regulated cytoskeleton-associated protein (Arc; also known as Arg3.1) is an immediate early gene product that is transcribed in dendritic spines and, to date, has been best characterized as a positive regulator of AMPAR endocytosis during long-term depression (LTD) through interaction with endocytic proteins. Here, we show that protein interacting with C terminal kinase 1 (PICK1), a protein known to bind to the GluA2 subunit of AMPARs and associated with AMPAR trafficking, was pulled-down from brain homogenates and synaptosomes when using Arc as immobilized bait. Fluctuation and FLIM-FRET-Phasor analysis revealed direct interaction between these proteins when co-expressed that was increased under depolarizing conditions in live cells. At the plasma membrane, Arc-mCherry oligomerization was found to be concentration dependent. Additionally, co-expression of Arc-mCherry and EGFP-PICK1 followed by depolarizing conditions resulted in significant increases in the number and size of puncta containing both proteins. Furthermore, we identified the Arc binding region to be the first 126 amino acids of the PICK1 BAR domain. Overall, our data support a novel interaction and model where PICK1 mediates Arc regulation of AMPARs particularly under depolarizing conditions.