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Fluorescence imaging of live cells allows for the observation of dynamic processes inside cells in real time. Here we describe a strategy to image clathrin-coated vesicle dynamics in a single focal plane at the trans-Golgi network of the yeast Saccharomyces cerevisiae. This method can be readily adapted for live cell imaging of a diverse set of dynamic processes within cells.

SummarySchwann cells respond to acute axon damage by transiently transdifferentiating into specialized repair cells that restore sensorimotor function. However, the molecular systems controlling repair cell formation and function are not well defined and consequently it is unclear whether this form of cellular plasticity has a role in peripheral neuropathies. Here we identify Mitf as a transcriptional sensor of axon damage under the control of Nrg-ErbB-PI3K-PI5K-mTorc2 signaling. Mitf regulates a core transcriptional program for generating functional repair Schwann cells following injury and during peripheral neuropathies caused by CMT4J and CMT4D. In the absence of Mitf, core genes for epithelial-to-mesenchymal transition, metabolism and dedifferentiation are misexpressed and nerve repair is disrupted. Taken together, our findings demonstrate that Schwann cells monitor axonal health using a phosphoinositide signaling system that controls Mitf, which is critical for activating cellular plasticity and counteracting neural disease.HighlightsMitf-induced Schwann cell plasticity is triggered by peripheral neuropathy.Nrg-ErbB signaling activates Mitf via cytoplasmic-to-nuclear translocation.Mitf restores sensorimotor function following axonal breakdown.Mitf regulates a core repair program across both injury and neurodegeneration.

Phosphoinositides serve as key membrane determinants for assembly of clathrin coat proteins that drive formation of clathrin-coated vesicles. At the trans-Golgi network (TGN), phosphatidylinositol 4-phosphate (PtdIns4P) plays important roles in recruitment of two major clathrin adaptors, Gga (Golgi-localized, gamma-adaptin ear homology, Arf-binding) proteins and the AP-1 (assembly protein-1) complex. The molecular mechanisms that mediate localization of phosphatidylinositol kinases responsible for synthesis of PtdIns4P at the TGN are not well characterized. We identify two motifs in the yeast phosphatidylinositol 4-kinase, Pik1, which are required for binding to the VHS domain of Gga2. Mutations in these motifs that inhibit Gga2–VHS binding resulted in reduced Pik1 localization and delayed accumulation of PtdIns4P and recruitment of AP-1 to the TGN. The Pik1 homolog in mammals, PI4KIIIβ, interacted preferentially with the VHS domain of GGA2 compared with VHS domains of GGA1 and GGA3. Depletion of GGA2, but not GGA1 or GGA3, specifically affected PI4KIIIβ localization. These results reveal a conserved role for Gga proteins in regulating phosphatidylinositol 4-kinase function at the TGN.

Yeast Irc6p is a novel type of conserved clathrin coat accessory protein that functions in clathrin-mediated traffic between the trans-Golgi network and endosomes, linking clathrin adaptor complex AP-1 and the Rab GTPase Ypt31p. Irc6p and the mammalian homologue p34 are founding members of a new G protein–like family., Clathrin coat accessory proteins play key roles in transport mediated by clathrin-coated vesicles. Yeast Irc6p and the related mammalian p34 are putative clathrin accessory proteins that interact with clathrin adaptor complexes. We present evidence that Irc6p functions in clathrin-mediated traffic between the trans-Golgi network and endosomes, linking clathrin adaptor complex AP-1 and the Rab GTPase Ypt31p. The crystal structure of the Irc6p N-terminal domain revealed a G-protein fold most related to small G proteins of the Rab and Arf families. However, Irc6p lacks G-protein signature motifs and high-affinity GTP binding. Also, mutant Irc6p lacking candidate GTP-binding residues retained function. Mammalian p34 rescued growth defects in irc6∆ cells, indicating functional conservation, and modeling predicted a similar N-terminal fold in p34. Irc6p and p34 also contain functionally conserved C-terminal regions. Irc6p/p34-related proteins with the same two-part architecture are encoded in genomes of species as diverse as plants and humans. Together these results define Irc6p/p34 as a novel type of conserved clathrin accessory protein and founding members of a new G protein–like family.

Clathrin-coated vesicles mediate endocytosis and transport between the trans-Golgi network (TGN) and endosomes in eukaryotic cells. Clathrin adaptors play central roles in coat assembly, interacting with clathrin, cargo and membranes. Two main types of clathrin adaptor act in TGN-endosome traffic: GGA proteins and the AP-1 complex. Here we characterize the relationship between GGA proteins, AP-1 and other TGN clathrin adaptors using live-cell and super-resolution microscopy in yeast. We present evidence that GGA proteins and AP-1 are recruited sequentially in two waves of coat assembly at the TGN. Mutations that decrease phosphatidylinositol 4-phosphate (PtdIns(4)P) levels at the TGN slow or uncouple AP-1 coat assembly from GGA coat assembly. Conversely, enhanced PtdIns(4)P synthesis shortens the time between adaptor waves. Gga2p binds directly to the TGN PtdIns(4)-kinase Pik1p and contributes to Pik1p recruitment. These results identify a PtdIns(4)P-based mechanism for regulating progressive assembly of adaptor-specific clathrin coats at the TGN.

The evolutionarily conserved adaptor protein-3 (AP-3) complex mediates cargo-selective transport to lysosomes and lysosome-related organelles. To identify proteins that function in AP-3–mediated transport, we performed a genome-wide screen in Saccharomyces cerevisiae for defects in the vacuolar maturation of alkaline phosphatase (ALP), a cargo of the AP-3 pathway. Forty-nine gene deletion strains were identified that accumulated precursor ALP, many with established defects in vacuolar protein transport. Maturation of a vacuolar membrane protein delivered via a separate, clathrin-dependent pathway, was affected in all strains except those with deletions of YCK3, encoding a vacuolar type I casein kinase; SVP26, encoding an endoplasmic reticulum (ER) export receptor for ALP; and AP-3 subunit genes. Subcellular fractionation and fluorescence microscopy revealed ALP transport defects in yck3Δ cells. Characterization of svp26Δ cells revealed a role for Svp26p in ER export of only a subset of type II membrane proteins. Finally, ALP maturation kinetics in vac8Δ and vac17Δ cells suggests that vacuole inheritance is important for rapid generation of proteolytically active vacuolar compartments in daughter cells. We propose that the cargo-selective nature of the AP-3 pathway in yeast is achieved by AP-3 and Yck3p functioning in concert with machinery shared by other vacuolar transport pathways.