The endophilin curvature-sensitive motif requires electrostatic guidance to recycle synaptic vesicles in vivo

Curvature-sensing mechanisms assist proteins in executing particular actions on various membrane organelles. Here, we investigated the functional specificity of curvature-sensing amphipathic motifs through the study of endophilin, an endocytic protein for synaptic vesicle recycling. We generated chimeric endophilin proteins by replacing the endophilin amphipathic motif H0 with other curvature-sensing amphipathic motifs. We found that the role of amphipathic motifs cannot simply be extrapolated from the identity of their parental proteins. For example, the amphipathic motif of the nuclear pore complex protein NUP133 functionally replaced the synaptic role of endophilin H0. Interestingly, non-functional endophilin chimeras had similar defects — producing fewer synaptic vesicles but more endosomes — indicating that the curvature-sensing motifs in these chimeras have a common deficiency at reforming synaptic vesicles. Finally, we converted non-functional endophilin chimeras into functional proteins by changing the cationic property of amphipathic motifs, setting a precedent for reprogramming the functional specificity of curvature-sensing motifs in vivo.

Endocytic protein intersectin1-S shuttles into nucleus to suppress the DNA replication in breast cancer

Breast cancer is the most common type of cancer worldwide. However, the well-known molecular biomarkers are not enough to meet the needs of precision medicine. In search for novel targets in this regard, we reported ITSN1 (intersectin1) as one of the candidates through mRNA microarray analysis. In the present study, we reported that endocytic protein ITSN1-S exists not only in the cytoplasm but also in nuclei of breast cancer cells. ITSN1-S′ functional nuclear localization signal is within its residues 306–312. Its nuclear export signal (NES) resides within its SH3 domains. We also found, the interaction between the CC domain of nuclear ITSN1-S and the NT domain of nuclear DNA helicase II (NDH II) directly suppressed the DNA replication and nascent DNA synthesis by inhibiting the R-loops resolution in breast cancer cells. Furthermore, the interaction between the EH domains of cytoplasmic ITSN1-S and PI3KC2α inhibit cell migration and invasion by inactivating the PI3KC2α-AKT pathway. Our results were confirmed in both ITSN1 gene knockout cells and in vivo assays. Finally, our clinical data showed a potential application of the combined consideration of the cytoplasmic and nuclear ITSN1-S as an independent prognosis factor. In conclusion, our study revealed ITSN1-S′ novel positioning in the nuclei of breast cancer cells, its function in suppressing DNA replication, and its potential application in improved breast cancer prognosis.

Microtubule assembly by soluble tau impairs vesicle endocytosis and excitatory neurotransmission via dynamin sequestration in Alzheimer's disease synapse model

Elevation of soluble wild-type (WT) tau occurs in synaptic compartments in Alzheimer's disease. We addressed whether tau elevation affects synaptic transmission at the calyx of Held in brainstem slices. Whole-cell loading of WT human tau (h-tau) in presynaptic terminals at 10-20 μM caused microtubule (MT) assembly and activity-dependent rundown of excitatory neurotransmission. Capacitance measurements revealed that the primary target of WT h-tau is vesicle endocytosis. Blocking MT assembly using nocodazole prevented tau-induced impairments of endocytosis and neurotransmission. Immunofluorescence imaging analyses revealed that MT assembly by WT h-tau loading was associated with an increased bound fraction of the endocytic protein dynamin. A synthetic dodecapeptide corresponding to dynamin-1-pleckstrin-homology domain inhibited MT-dynamin interaction and rescued tau-induced impairments of endocytosis and neurotransmission. We conclude that elevation of presynaptic WT tau induces de novo assembly of MTs, thereby sequestering free dynamins. As a result, endocytosis and subsequent vesicle replenishment are impaired, causing activity-dependent rundown of neurotransmission.

Light-dependent inhibition of clathrin-mediated endocytosis in yeast

Clathrin-mediated endocytosis (CME) is an essential cellular process, which is evolutionarily conserved among eukaryotes. Yeast constitutes a powerful genetic model to dissect the complex endocytic machinery, yet there is a lack of pharmacological agents that could complement genetics in selectively and reversibly interfere with CME in these organisms. TL2 is a light-regulated peptide inhibitor that targets the AP2/β-arrestin interaction and that can photocontrol CME with high spatiotemporal precision in mammalian cells. Here, we study endocytic protein dynamics by live-cell imaging of the fluorescently tagged coat-associated protein Sla1-GFP and demonstrate that TL2 retains its inhibitory activity in S. cerevisiae spheroplasts, thus providing a unique tool for acute and reversible CME modulation in yeast.

The C2 domain of the ubiquitin ligase Rsp5 is required for ubiquitination of the endocytic protein Rvs167 upon change of nitrogen source

ABSTRACT Ubiquitination is a key signal for endocytosis of proteins on the plasma membrane. The ubiquitin ligase Rsp5 of Saccharomyces cerevisiae, which contains an amino-terminal membrane-binding C2 domain, three substrate-recognizing tryptophan-tryptophan (WW) domains and a carboxyl-terminal catalytic homologous to the E6-AP carboxyl terminus (HECT) domain, can ubiquitinate plasma membrane proteins directing them for endocytosis. Here, we examined the roles of the C2 domain in endocytosis for the downregulation of the general amino acid permease Gap1, which is one of nitrogen-regulated permeases in S. cerevisiae. First, we constructed several rsp5 mutants producing Rsp5 variants without the C2 domain or with amino acid changes of membrane-binding lysine residues. These mutants showed defects in endocytosis of Gap1 in response to a preferred nitrogen source. Intriguingly, we found that ubiquitination of Gap1 in these mutant cells was highly similar to that in wild-type cells during endocytosis. These results indicate that the C2 domain is essential for endocytosis but not for ubiquitination of substrates such as Gap1. Moreover, genetic and biochemical analyses showed that the endocytic protein Rvs167 was ubiquitinated via Rsp5 and the C2 domain was required for efficient ubiquitination in response to a preferred nitrogen source. Here, we propose a mechanism for the C2 domain-mediated endocytosis of plasma membrane permeases.

Fluctuation Imaging of LRRK2 Reveals that the G2019S Mutation Alters Spatial and Membrane Dynamics

Mutations within the Leucine-Rich Repeat Kinase 2 (LRRK2) gene are the most common genetic cause of autosomal and sporadic Parkinson’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—all of which could lead to neurodegeneration associated with PD.