Essential function of transmembrane transcription factor MYRF in promoting transcription of miRNA lin-4 during C. elegans development.

Precise developmental timing control is essential for organism formation and function, but its mechanisms are unclear. In C. elegans, the microRNA lin-4 critically regulates developmental timing by post-transcriptionally downregulating the larval-stage-fate controller LIN-14. However, the mechanisms triggering the activation of lin-4 expression toward the end of the first larval stage remain unknown. We demonstrate that the transmembrane transcription factor MYRF-1 is necessary for lin-4 activation. MYRF-1 is initially localized on the cell membrane, and its increased cleavage and nuclear accumulation coincide with lin-4 expression timing. MYRF-1 regulates lin-4 expression cell-autonomously and hyperactive MYRF-1 can prematurely drive lin-4 expression in embryos and young first-stage larvae. The tandem lin-4 promoter DNA recruits MYRF-1GFP to form visible loci in the nucleus, suggesting that MYRF-1 directly binds to the lin-4 promoter. Our findings identify a crucial link in understanding developmental timing regulation and establish MYRF-1 as a key regulator of lin-4 expression.

MYRF: A unique transmembrane transcription factor- from proteolytic self-processing to its multifaceted roles in animal development.

The Myelin Regulator Factor (MYRF) is a master regulator governing myelin formation and maintenance in the central nervous system. The conservation of MYRF across metazoans and its broad tissue expression suggest it has functions extending beyond the well-established role in myelination. Loss of MYRF results in developmental lethality in both invertebrates and vertebrates, and MYRF haploinsufficiency in humans causes MYRF-related Cardiac Urogenital Syndrome, underscoring its importance in animal development; however, these mechanisms are largely unexplored. MYRF, an unconventional transcription factor, begins embedded in the membrane and undergoes intramolecular chaperone mediated trimerization, which triggers self-cleavage, allowing its N-terminal segment with an Ig-fold DNA-binding domain to enter the nucleus for transcriptional regulation. Recent research suggests developmental regulation of cleavage, yet the mechanisms remain enigmatic. While some parts of MYRF's structure have been elucidated, others remain obscure, leaving questions about how these motifs are linked to its intricate processing and function.

Actin assembly at sites of contact between the cortical ER and endocytic pits promotes ER autophagy.

A recent screen of the Saccharomyces cerevisiae deletion library implicated End3 in autophagy of the endoplasmic reticulum (ER). Together with Pan1, End3 coordinates endocytic site initiation with the localized assembly of branching actin filaments that promotes invagination of endocytic pits. Oxysterol binding proteins function as an inter-organelle bridge by interacting with VAP proteins on the cortical ER and type I myosins on the endocytic pit. These proteins not only promote localized actin assembly at contact sites, they are required for ER autophagy as well. We propose that localized actin polymerization can push the edge of an ER sheet from the cell cortex toward the site of autophagosome assembly near the vacuole.

The LRR-TM protein PAN-1 interacts with MYRF to promote its nuclear translocation in synaptic remodeling.

Neural circuits develop through a plastic phase orchestrated by genetic programs and environmental signals. We have identified a leucine-rich-repeat domain transmembrane protein PAN-1 as a factor required for synaptic rewiring in C. elegans. PAN-1 localizes on cell membrane and binds with MYRF, a membrane-bound transcription factor indispensable for promoting synaptic rewiring. Full-length MYRF was known to undergo self-cleavage on ER membrane and release its transcriptional N-terminal fragment in cultured cells. We surprisingly find that MYRF trafficking to cell membrane before cleavage is pivotal for C. elegans development and the timing of N-MYRF release coincides with the onset of synaptic rewiring. On cell membrane PAN-1 and MYRF interact with each other via their extracellular regions. Loss of PAN-1 abolishes MYRF cell membrane localization, consequently blocking myrf-dependent neuronal rewiring process. Thus, through interactions with a cooperating factor on the cell membrane, MYRF may link cell surface activities to transcriptional cascades required for development.

Characterization of PAN-1, a Carbapenem-Hydrolyzing Class B ß-Lactamase From the Environmental Gram-Negative Pseudobacteriovorax antillogorgiicola.

The gene encoding the metallo-ß-lactamase (MßL) PAN-1 was identified in the genome of the environmental Gram-negative species Pseudobacteriovorax antillogorgiicola. PAN-1 shares 57% amino-acid identity with the acquired MßL SPM-1, its closest relative. Kinetic parameters performed on purified PAN-1 showed it displayed a hydrolytic activity toward most ß-lactams including carbapenems but spared cefepime and aztreonam. These results further highlight that environmental bacterial species may be reservoirs of MßL encoding genes.

Target of rapamycin complex 2-dependent phosphorylation of the coat protein Pan1 by Akl1 controls endocytosis dynamics in Saccharomyces cerevisiae.

Target of rapamycin complex 2 (TORC2) is a widely conserved serine/threonine protein kinase. In the yeast Saccharomyces cerevisiae, TORC2 is essential, playing a key role in plasma membrane homeostasis. In this role, TORC2 regulates diverse processes, including sphingolipid synthesis, glycerol production and efflux, polarization of the actin cytoskeleton, and endocytosis. The major direct substrate of TORC2 is the AGC-family kinase Ypk1. Ypk1 connects TORC2 signaling to actin polarization and to endocytosis via the flippase kinases Fpk1 and Fpk2. Here, we report that Fpk1 mediates TORC2 signaling to control actin polarization, but not endocytosis, via aminophospholipid flippases. To search for specific targets of these flippase kinases, we exploited the fact that Fpk1 prefers to phosphorylate Ser residues within the sequence RXS(L/Y)(D/E), which is present ∼90 times in the yeast proteome. We observed that 25 of these sequences are phosphorylated by Fpk1 in vitro We focused on one sequence hit, the Ark/Prk-family kinase Akl1, as this kinase previously has been implicated in endocytosis. Using a potent ATP-competitive small molecule, CMB4563, to preferentially inhibit TORC2, we found that Fpk1-mediated Akl1 phosphorylation inhibits Akl1 activity, which, in turn, reduces phosphorylation of Pan1 and of other endocytic coat proteins and ultimately contributes to a slowing of endocytosis kinetics. These results indicate that the regulation of actin polarization and endocytosis downstream of TORC2 is signaled through separate pathways that bifurcate at the level of the flippase kinases.

A Global Analysis of Kinase Function in Candida albicans Hyphal Morphogenesis Reveals a Role for the Endocytosis Regulator Akl1.

The human pathogenic fungus Candida albicans can switch between yeast and hyphal morphologies as a function of environmental conditions and cellular physiology. The yeast-to-hyphae morphogenetic switch is activated by well-established, kinase-based signal transduction pathways that are induced by extracellular stimuli. In order to identify possible inhibitory pathways of the yeast-to-hyphae transition, we interrogated a collection of C. albicans protein kinases and phosphatases ectopically expressed under the regulation of the TETon promoter. Proportionately more phosphatases than kinases were identified that inhibited hyphal morphogenesis, consistent with the known role of protein phosphorylation in hyphal induction. Among the kinases, we identified AKL1 as a gene that significantly suppressed hyphal morphogenesis in serum. Akl1 specifically affected hyphal elongation rather than initiation: overexpression of AKL1 repressed hyphal growth, and deletion of AKL1 resulted in acceleration of the rate of hyphal elongation. Akl1 suppressed fluid-phase endocytosis, probably via Pan1, a putative clathrin-mediated endocytosis scaffolding protein. In the absence of Akl1, the Pan1 patches were delocalized from the sub-apical region, and fluid-phase endocytosis was intensified. These results underscore the requirement of an active endocytic pathway for hyphal morphogenesis. Furthermore, these results suggest that under standard conditions, endocytosis is rate-limiting for hyphal elongation.

Switch-like Arp2/3 activation upon WASP and WIP recruitment to an apparent threshold level by multivalent linker proteins in vivo.

Actin-related protein 2/3 (Arp2/3) complex activation by nucleation promoting factors (NPFs) such as WASP, plays an important role in many actin-mediated cellular processes. In yeast, Arp2/3-mediated actin filament assembly drives endocytic membrane invagination and vesicle scission. Here we used genetics and quantitative live-cell imaging to probe the mechanisms that concentrate NPFs at endocytic sites, and to investigate how NPFs regulate actin assembly onset. Our results demonstrate that SH3 (Src homology 3) domain-PRM (proline-rich motif) interactions involving multivalent linker proteins play central roles in concentrating NPFs at endocytic sites. Quantitative imaging suggested that productive actin assembly initiation is tightly coupled to accumulation of threshold levels of WASP and WIP, but not to recruitment kinetics or release of autoinhibition. These studies provide evidence that WASP and WIP play central roles in establishment of a robust multivalent SH3 domain-PRM network in vivo, giving actin assembly onset at endocytic sites a switch-like behavior.

Targeted disruption of an EH-domain protein endocytic complex, Pan1-End3.

Pan1 is a multi-domain scaffold that enables dynamic interactions with both structural and regulatory components of the endocytic pathway. Pan1 is composed of Eps15 Homology (EH) domains which interact with adaptor proteins, a central region that is responsible for its oligomerization and C-terminal binding sites for Arp2/3, F-actin, and type-I myosin motors. In this study, we have characterized the binding sites between Pan1 and its constitutive binding partner End3, another EH domain containing endocytic protein. The C-terminal End3 Repeats of End3 associate with the N-terminal part of Pan1's central coiled-coil region. These repeats appear to act independently of one another as tandem, redundant binding sites for Pan1. The end3-1 allele was sequenced, and corresponds to a C-terminal truncation lacking the End3 Repeats. Mutations of the End3 Repeats highlight that those residues which are identical between these repeats serve as contact sites for the interaction with Pan1.

Pan1 is an intrinsically disordered protein with homotypic interactions.

The yeast scaffold protein Pan1 contains two EH domains at its N-terminus, a predicted coiled-coil central region, and a C-terminal proline-rich domain. Pan1 is also predicted to contain regions of intrinsic disorder, characteristic of proteins that have many binding partners. In vitro biochemical data suggest that Pan1 exists as a dimer, and we have identified amino acids 705 to 848 as critical for this homotypic interaction. Tryptophan fluorescence was used to further characterize Pan1 conformational states. Pan1 contains four endogenous tryptophans, each in a distinct region of the protein: Trp(312) and Trp(642) are each in an EH domain, Trp(957) is in the central region, and Trp(1280) is a critical residue in the Arp2/3 activation domain. To examine the local environment of each of these tryptophans, three of the four tryptophans were mutagenized to phenylalanine to create four proteins, each with only one tryptophan residue. When quenched with acrylamide, these single tryptophan mutants appeared to undergo collisional quenching exclusively and were moderately accessible to the acrylamide molecule. Quenching with iodide or cesium, however, revealed different Stern-Volmer constants due to unique electrostatic environments of the tryptophan residues. Time-resolved fluorescence anisotropy data confirmed structural and disorder predictions of Pan1. Further experimentation to fully develop a model of Pan1 conformational dynamics will assist in a deeper understanding of the mechanisms of endocytosis.