InteractiveFly: GeneBrief

Putative Achaete Scute Target 1: Biological Overview | References

Gene name - Putative Achaete Scute Target 1

Synonyms -

Cytological map position - 87C6-87C6

Function - enzyme

Keywords - plasma membrane protein that genetically interacts with Notch, contributes to endocytosis and differentiation of photoreceptors R1/R6/R7 and cone cells, required for assembly of the subsynaptic muscle membrane reticulum at the neuromuscular junction

Symbol - Past1

FlyBase ID: FBgn0016693

Genetic map position - chr3R:12,697,328-12,704,881

NCBI classification - Eps15 homology domain (EHD), C-terminal domain - EF-Hand 1, calcium-binding site - Dynamin superfamily - P-loop containing nucleoside triphosphate hydrolase

Cellular location - surface transmembrane

NCBI link: EntrezGene, Nucleotide, Protein
Past1 orthologs: Biolitmine
Recent literature
Okada, A. K., Teranishi, K., Ambroso, M. R., Isas, J. M., Vazquez-Sarandeses, E., Lee, J. Y., Melo, A. A., Pandey, P., Merken, D., Berndt, L., Lammers, M., Daumke, O., Chang, K., Haworth, I. S. and Langen, R. (2021). Lysine acetylation regulates the interaction between proteins and membranes. Nat Commun 12(1): 6466. PubMed ID: 34753925
Lysine acetylation regulates the function of soluble proteins in vivo, yet it remains largely unexplored whether lysine acetylation regulates membrane protein function. Thia study use bioinformatics, biophysical analysis of recombinant proteins, live-cell fluorescent imaging and genetic manipulation of Drosophila to explore lysine acetylation in peripheral membrane proteins. Analysis of 50 peripheral membrane proteins harboring BAR, PX, C2, or EHD membrane-binding domains reveals that lysine acetylation predominates in membrane-interaction regions. Acetylation and acetylation-mimicking mutations in three test proteins, amphiphysin, EHD2, and synaptotagmin1, strongly reduce membrane binding affinity, attenuate membrane remodeling in vitro and alter subcellular localization. This effect is likely due to the loss of positive charge, which weakens interactions with negatively charged membranes. In Drosophila, acetylation-mimicking mutations of amphiphysin cause severe disruption of T-tubule organization and yield a flightless phenotype. These data provide mechanistic insights into how lysine acetylation regulates membrane protein function, potentially impacting a plethora of membrane-related processes.

Membranes form elaborate structures that are highly tailored to their specialized cellular functions, yet the mechanisms by which these structures are shaped remain poorly understood. This study shows that the conserved membrane-remodeling C-terminal Eps15 Homology Domain (EHD) protein Past1 is required for the normal assembly of the subsynaptic muscle membrane reticulum (SSR) at the Drosophila melanogaster larval neuromuscular junction (NMJ). past1 mutants exhibit altered NMJ morphology, decreased synaptic transmission, reduced glutamate receptor levels, and a deficit in synaptic homeostasis. The membrane-remodeling proteins Amphiphysin and Syndapin colocalize with Past1 in distinct SSR subdomains, and collapse into Amphiphysin-dependent membrane nodules in the SSR of past1 mutants. These results suggest a mechanism by which the coordinated actions of multiple lipid-binding proteins lead to the elaboration of increasing layers of the SSR, and uncover new roles for an EHD protein at synapses (Koles, 2015).

Dozens of lipid-binding proteins dynamically remodel membranes, generating diverse cell shapes, sculpting organelles, and promoting traffic between subcellular compartments. Although the activities of many of these membrane-remodeling proteins have been studied individually, what is lacking is an understanding of how membrane-remodeling factors work together to generate specialized membranes in vivo (Koles, 2015).

C-terminal Eps15 Homology Domain (EHD)-family proteins encode large membrane-binding ATPases with structural similarity to dynamin and function at a variety of steps of membrane transport (Naslavsky, 2011). These proteins contain an ATPase domain, a helical lipid-binding domain, and a carboxy-terminal EH domain that interacts with Asn-Pro-Phe (NPF)-containing binding partners (Naslavsky, 2011). Although their mechanism of action is not fully understood, it is postulated that C-terminal EHD proteins bind and oligomerize in an ATP-dependent manner on membrane compartments, where they are involved in the trafficking of cargo. The mouse and human genomes each contain four highly similar EHD proteins (EHD1-4), which have both unique and overlapping functions (Naslavsky, 2011). EHD proteins interact with several members of the Bin/Amphiphysin/Rvs167 (BAR) and Fes/Cip4 homology-BAR (F-BAR) protein families, which themselves can remodel membranes via their crescent-shaped dimeric BAR domains (Masuda, 2010). In mammals, EHD proteins associate with the NPF motifs of the F-BAR proteins Syndapin I and II, and these interactions are critical for recycling of cargo from endosomes to the plasma membrane in cultured cells. In Caenorhabditis elegans, the sole EHD protein Rme-1 colocalizes and functions with the BAR protein Amphiphysin and the F-BAR protein Syndapin, also via their NPF motifs (Pant, 2009). Further, EHD1 has been suggested to drive the scission of endosomal recycling tubules generated by the membrane-deforming activities of Syndapin 2 and another NPF-containing protein, MICAL-L1. However, the combined membrane-remodeling activities that might arise in vivo from the shared functions of C-terminal EHD and NPF-containing proteins remain unclear (Koles, 2015).

The Drosophila neuromuscular junction (NMJ) is a powerful system in which to study membrane remodeling. On the postsynaptic side of the NMJ, a highly convoluted array of muscle membrane infoldings called the subsynaptic reticulum (SSR) incorporates neurotransmitter receptors, ion channels, and cell adhesion molecules. Assembly of the SSR during larval growth involves activity-dependent targeted exocytosis mediated by the small GTPase Ral and its effector, the exocyst complex (Teodoro, 2013), as well as the t-SNARE (target soluble N-ethylmaleimide-sensitive factor attachment protein receptor) receptor gtaxin/Syx18 (Gorczyca, 2007) and scaffolding proteins such as Discs Large (Dlg). Many proteins with predicted membrane-remodeling activities, including Drosophila homologues of Syndapin (Synd) and Amphiphysin (Amph), localize extensively to SSR membranes, making them prime candidates to facilitate SSR elaboration. Amph regulates the postsynaptic turnover of the trans-synaptic cell adhesion molecule FasII, but its role in organizing the SSR is unknown (Koles, 2015).

The Drosophila melanogaster genome encodes a single C-terminal EHD protein called Putative achaete/scute target (Past1). Past1 mutants exhibit defects in endocytic recycling in larval nephrocytes, sterility and aberrant development of the germline, and short lifespan (Olswang-Kutz, 2009), but the functions of Past1 at the NMJ have not been explored. Mammalian EHD1 localizes to the mouse NMJ, but its function there has been difficult to ascertain, perhaps due to redundancy with other EHD proteins. This study takes advantage of the fact that Past1 encodes the only Drosophila C-terminal EHD protein and define its role at the NMJ (Koles, 2015).

Putting together the current observations at the NMJ and in S2 cells with previous results from other groups, a new working model is proposed for how Past1 functions in synaptic membrane elaboration. The first key observation is that Past1 is required for normal elaboration of the SSR and that this function depends on its ATP-binding and thus membrane-remodeling activity. Next, in wild-type SSR, Amph was found to localize to a domain proximal to the bouton, whereas Past1 and Synd localize to a more extended tubulovesicular domain. By contrast, in the absence of Past1, the SSR rearranges into highly organized subdomains, with a core of Synd surrounded by a shell of Amph (likely corresponding to membrane sheets. Amph was found to be required for the formation of the sheets (perhaps by regulating the tight curvature at the tips of these membrane structures) and for consolidation of Synd into nodules. Further, FRAP data indicate that the nodules result in significantly increased membrane flow within the SSR relative to wild-type SSR, suggesting reduced complexity. Finally, S2 cell data indicate that Past1 may activate the membrane-binding/remodeling activity of Synd (Koles, 2015).

These results suggest a novel mechanism for SSR elaboration at the wild-type NMJ involving sequential steps of membrane remodeling. In this model, Amph localizes and generates membrane tubules proximal to the bouton, and Past1 and Synd work together to further elaborate the tubules distal to the bouton. Successive rounds of these events could lead to the growth and expansion of layers of reticulum. In past1 mutants, this process is severely compromised, resulting in nodules containing a core of inactive Synd packed by Amph-dependent membrane sheets (Koles, 2015).

One issue that remains to be resolved is whether direct physical interactions among Past1, Amph, and Synd (within the SSR subdomains to which they colocalize) contribute to Past1-dependent membrane remodeling at the NMJ, as they do in other systems (Xu, 2004; Braun, 2005; Pant, 2009). S2 cell data suggest that Past1 and Synd functionally interact in vivo. However, no Past1-Amph or Past1-Synd complexes were detected using coprecipitation experiments in extracts from Drosophila larvae or S2 cells or with purified proteins, suggesting that either they do not directly interact or their interactions are not preserved in solution under the conditions tested. Genetic experiments at the NMJ using mutations that disrupt putative Past1-Synd and Past1-Amph interactions are unlikely to be informative because synd and amph single mutants exhibit no dramatic phenotype in SSR organization, perhaps due to redundancy with other membrane-remodeling proteins. In fact, in addition to Amph and Synd, it was found that the BAR proteins Cip4 and dRich are localized to nodules in past1 mutants, suggesting that multiple membrane-remodeling proteins are available to function in the Past1-dependent pathway. In the future, it will be important to build into a working model the additional roles of these and other SSR-localized membrane-remodeling proteins, as well as the timing of exocyst-dependent membrane addition (Teodoro, 2013; Koles, 2015 and references therein).

The results demonstrate that postsynaptic Past1 plays critical roles in the structure and function of the Drosophila NMJ. Past1 mutant NMJs exhibit aberrant morphology and excess ghost boutons. These ghost boutons are unlikely to be due to defective clearance of excess neuronal membrane, since large amounts of neuronal debris were not observed. They are also unlikely to be related to excess ghost boutons seen in Wingless (Wg) signaling pathway mutants, since past1 mutants do not phenocopy many other aspects of reduced Wg signaling, including increased GluR levels, disrupted presynaptic function, and reduction in bouton number. The likeliest interpretation is that Past1 functions directly in SSR membrane elaboration, consistent with EM observations, and ghost boutons may arise when membrane nodules become too severe to allow SSR assembly around boutons that form toward the end of larval development (Koles, 2015).

Another prominent synaptic phenotype that found in past1 mutants is a strong and specific reduction in localization of GluRIIA to postsynaptic specializations, resulting in decreased mEPSP amplitude. This decrease in GluRIIA could potentially arise by many mechanisms, including altered transcriptional or translational regulation or GluR traffic to or from the synapse. Indeed, expression of a dominant-negative EHD1 suppresses AMPA glutamate receptor recycling in hippocampal dendritic spines. Although there has been little evidence that Drosophila GluRs are regulated by membrane traffic, the data implicating the membrane-remodeling protein Past1 indicate that this may be the case. Finally, unlike the great majority of perturbations that reduce GluRIIA levels, past1 mutants surprisingly fail to compensate for this loss by homeostatic up-regulation of presynaptic release, suggesting that Past1 could be involved in relaying an as-yet-unidentified retrograde signal for synaptic homeostasis. Further work exploring mechanisms of GluRIIA regulation and retrograde signaling will be required to understand the role of Past1 in these events (Koles, 2015).

The present data cannot distinguish whether the function of Past1 in GluR traffic or homeostasis is directly related to its role in SSR elaboration, and it is possible that membrane compartments independent of the SSR are required for these functions and are disrupted in the mutant. The finding that GluRIIA levels are still reduced in amph; past1 double mutants although SSR nodules are suppressed supports the conclusion that GluR localization defects are independent of aberrant SSR morphogenesis. Of note, many mutants with severely defective SSR and/or reduced GluR levels exhibit normal homeostasis (e.g., GluRIIA, which also has reduced SSR, Dlg, Gtaxin, and Pak1, suggesting that homeostasis is a specific function of Past1 rather than a general SSR- or GluR-related defect (Koles, 2015).

Past1 represents the sole EHD homologue in Drosophila, whereas mammals express four EHD proteins with distinct functions (Naslavsky, 2011). Of importance, many of the roles for EHD proteins at the NMJ and in muscle are likely to be conserved. Past1 localizes to the NMJ, the muscle cortex, and myotendinous junctions. However, unlike EHD1, Past1 does not significantly localize to t-tubules. The activities identified for Past1 at the Drosophila NMJ may inform mechanisms by which EHD2 participates in sarcolemmal repair at the muscle cortex (Marg, 2012), EHD3 functions in cardiac muscle physiology (Curran, 2014), and EHD1 and EHD4 act at the mouse NMJ (Mate, 2012). The current findings set the stage for uncovering how neuromuscular synapses are formed and elaborated and illustrate how cooperation between lipid-remodeling proteins can create highly complex membrane structures (Koles, 2015).

Past1 modulates Drosophila eye development

Endocytosis is a multi-step process involving a large number of proteins, both general factors, such as clathrin and adaptor protein complexes, and unique proteins, which modulate specialized endocytic processes, like the EHD proteins. EHDs are a family of Eps15 Homology Domain containing proteins that consists of four mammalian homologs, one C. elegans, one Drosophila melanogaster and two plants orthologs. These membrane-associated proteins are involved in different steps of endocytic trafficking pathways. The Drosophila EHD ortholog, PAST1, has been shown to associate predominantly with the plasma membrane. Mutations in Past1 result in defects in endocytosis, male sterility, temperature sensitivity and premature death of the flies. Also, Past1 genetically interacts with Notch. The present study investigated the role of PAST1 in the developing fly eye. In mutant flies lacking PAST1, abnormal differentiation of photoreceptors R1, R6 and R7 was evident, with partial penetrance. Likewise, five cone cells were present instead of four. Expression of transgenic PAST1 resulted in a dominant negative effect, with a phenotype similar to that of the deletion mutant, and appearance of additional inter-ommatidial pigment cells. These results strongly suggest a role for PAST1 in differentiation of photoreceptors R1/R6/R7 and cone cells of the fly ommatidia (Dorot, 2017).

In the Past1 null mutant flies no external eye abnormalities were observed, but the development of photoreceptors R1, R6 and R7, as well as of cone cells, was abnormal. In flies overexpressing PAST1 in the eye external abnormalities were noted, as well as an abnormal number of photoreceptors, cone cells and IPCs. Defects in ommatidium development accompanied by no external abnormalities in the eye have been previously reported for other mutations. For example, the external eye of mutants in Muscleblind (Mbl), a regulator of alternative splicing, appear to be normal, yet tangential sections revealed that they harbored ommatidia defects. Another example involves Crumbs (Crb), a transmembrane protein which is essential for biogenesis of adherens junctions and for establishing apical-basal polarity in Drosophila epithelia by downregulating endocytosis of Notch and Delta during eye development. External morphology of mutant Crb (crbS87-2) was reported to be normal. However, internal structure of adult CrbS87-2 mutant ommatidia was found to be defective with shortened and bulkier rhabdomeres, often in contact with each other rather than being distinct as in wild type eyes (Dorot, 2017).

Interestingly in Past1 null flies, the ommatidia always contained at least one R7 photoreceptor, with some having two R7 photoreceptors with a concomitant loss of either R1 or R6 or both. These results imply that differentiation to R1 or R6 was abrogated and instead, at least one R7 photoreceptor was developed. No change in Boss staining was detected in R7 cells of eye discs from Past1 null mutants or from transgenic GFP-PAST1 flies, while in Past1 null mutants the ommatidia always contained at least four cone cells and some ommatidia had five cone cells (Dorot, 2017).

Previously work has shown that Past1 genetically interacts with Notch in the wing (Olswang-Kutz, 2009). The results of the present study indicated that Past1 plays a role during eye development, which is known to be regulated by Notch. Interestingly, endocytosis of Notch seemed abnormal in early-mid pupal mutant eyes. Thus, higher level of Notch staining was detected at the vicinity of the plasma membrane of mutant pigment cells, indicating abrogated endocytosis. This result implies abnormal internalization of Notch in the absence of PAST1 and hints to the possibility that PAST1 modulates internalization of Notch during eye development. (Dorot, 2017).

The results well fit the model suggested by Tomlinson (2011) for the development of photoreceptors R1/R6/R7 and cone cells. According to this model Notch overexpression in either R1 or R6 photoreceptors, leads to development of one of these cells into an extra R7 photoreceptor. Additionally, overexpression of Notch in R1 or R6 combined with an absence of Sev leads to the appearance of an extra cone cell. It is proposed that lack of PAST1 elevates Notch activation in R1 and R6 photoreceptors, which subsequently develop into R7 or into cone cells. It is hypothesized that PAST1 negatively regulates Notch signaling in R1 and R6. Since penetrance of Past1 mutation in the eye is low, PAST1 is, most probably, not a major regulator of Notch signaling, but fine-tunes it. Further experiments are needed to establish the role of PAST1 in endocytosis of Notch (Dorot, 2017).

Eyes of flies overexpressing PAST1 were rough with regions of fused ommatidia and exhibited extra IPCs. Mutations that abrogate the normal cell death process of IPCs have been shown to lead to rough eyes. More so, overexpression of Notch (NFL or NICD) in the pupal retina led to appearance of extra IPCs. Therefore, it is assumed that the rough eyes phenotype in the PAST1 transgenic flies is due to abnormal PCD of IPCs (Dorot, 2017).

This study examined the ability of Garland cells overexpressing PAST1 to internalize fluorescently labeled Texas-Red avidin, as was examined for Past1 mutants. Garland cells overexpressing Past1 displayed attenuated endocytosis. Overexpression of PAST1 had a dominant negative effect, showing approximately 50-60% attenuation in endocytosis. A similar dominant negative effect has also been previously shown in HeLa cells overexpressing the mammalian EHD2, leading to attenuation of plasma membrane internalization (Dorot, 2017).

In conclusion, the results presented in this work highlight the importance of PAST1 for development of R1,R6 and R7 photoreceptors of the fly ommatidia (Dorot, 2017).

Drosophila Past1 is involved in endocytosis and is required for germline development and survival of the adult fly

Endocytosis, a key process in eukaryotic cells, has a central role in maintaining cellular homeostasis, nutrient uptake, development and downregulation of signal transduction. This complex process depends on several protein-protein interactions mediated by specific modules. One such module is the EH domain. The EH-domain-containing proteins comprise a family that includes four vertebrate members (EHD1-EHD4) and one Drosophila ortholog, Past1. This study used Drosophila as a model to understand the physiological role of this family of proteins. The two predicted Past1 transcripts are differentially expressed both temporally and spatially during the life cycle of the fly. Endogenous Past1 as well as Past1A and Past1B, expressed from plasmids, were localized mainly to the membrane of Drosophila-derived cells. Mutants were generated in the Past1 gene by excising a P-element inserted in it. The Past1 mutants reached adulthood but died precociously. They were temperature sensitive and infertile because of lesions in the reproductive system. Garland cells that originated from Past1 mutants exhibited a marked decrease in their ability to endocytose fluorescently labeled avidin. Genetic interaction was found between Past1 and members of the Notch signaling pathway, suggesting a role for Past1 in this developmentally crucial signaling pathway (Olswang-Kutz, 2009).

Functions of Pant1 orthologs in other species

Structural insights into the activation mechanism of dynamin-like EHD ATPases

Eps15 (epidermal growth factor receptor pathway substrate 15)-homology domain containing proteins (EHDs) comprise a family of dynamin-related mechano-chemical ATPases involved in cellular membrane trafficking. Previous studies have revealed the structure of the EHD2 dimer, but the molecular mechanisms of membrane recruitment and assembly have remained obscure. This study determined the crystal structure of an amino-terminally truncated EHD4 dimer. Compared with the EHD2 structure, the helical domains are 50 degrees rotated relative to the GTPase domain. Using electron paramagnetic spin resonance (EPR), this rotation was shown to align the two membrane-binding regions in the helical domain toward the lipid bilayer, allowing membrane interaction. A loop rearrangement in GTPase domain creates a new interface for oligomer formation. These results suggest that the EHD4 structure represents the active EHD conformation, whereas the EHD2 structure is autoinhibited, and reveal a complex series of domain rearrangements accompanying activation. A comparison with other peripheral membrane proteins elucidates common and specific features of this activation mechanism (Melo, 2017).

EHD proteins cooperate to generate caveolar clusters and to maintain caveolae during repeated mechanical stress

Caveolae introduce flask-shaped convolutions into the plasma membrane and help to protect the plasma membrane from damage under stretch forces. The protein components that form the bulb of caveolae are increasingly well characterized, but less is known about the contribution of proteins that localize to the constricted neck. This study made extensive use of multiple CRISPR/Cas9-generated gene knockout and knockin cell lines to investigate the role of Eps15 Homology Domain (EHD) proteins at the neck of caveolae. EHD1, EHD2, and EHD4 were shown to be recruited to caveolae. Recruitment of the other EHDs increases markedly when EHD2, which has been previously detected at caveolae, is absent. Construction of knockout cell lines lacking EHDs 1, 2, and 4 confirms this apparent functional redundancy. Two striking sets of phenotypes are observed in EHD1,2,4 knockout cells: (1) the characteristic clustering of caveolae into higher-order assemblies is absent; and (2) when the EHD1,2,4 knockout cells are subjected to prolonged cycles of stretch forces, caveolae are destabilized and the plasma membrane is prone to rupture. These data identify the first molecular components that act to cluster caveolae into a membrane ultrastructure with the potential to extend stretch-buffering capacity and support a revised model for the function of EHDs at the caveolar neck (Yeow, 2017).

PICK1 interacts with PACSIN to regulate AMPA receptor internalization and cerebellar long-term depression

The dynamic trafficking of AMPA receptors (AMPARs) into and out of synapses is crucial for synaptic transmission, plasticity, learning, and memory. The protein interacting with C-kinase 1 (PICK1) directly interacts with GluA2/3 subunits of the AMPARs. Although the role of PICK1 in regulating AMPAR trafficking and multiple forms of synaptic plasticity is known, the exact molecular mechanisms underlying this process remain unclear. This study reports a unique interaction between PICK1 and all three members of the protein kinase C and casein kinase II substrate in neurons (PACSIN) family and shows that they form a complex with AMPARs. These results reveal that knockdown of the neuronal-specific protein, PACSIN1, leads to a significant reduction in AMPAR internalization following the activation of NMDA receptors in hippocampal neurons. The interaction between PICK1 and PACSIN1 is regulated by PACSIN1 phosphorylation within the variable region and is required for AMPAR endocytosis. Similarly, the binding of PICK1 to the ubiquitously expressed PACSIN2 is also regulated by the homologous phosphorylation sites within the PACSIN2-variable region. Genetic deletion of PACSIN2, which is highly expressed in Purkinje cells, eliminates cerebellar long-term depression. This deficit can be fully rescued by overexpressing wild-type PACSIN2, but not by a PACSIN2 phosphomimetic mutant, which does not bind PICK1 efficiently. Taken together, these data demonstrate that the interaction of PICK1 and PACSIN is required for the activity-dependent internalization of AMPARs and for the expression of long-term depression in the cerebellum (Anggono, 2013).

Eps homology domain endosomal transport proteins differentially localize to the neuromuscular junction

Recycling of endosomes is important for trafficking and maintenance of proteins at the neuromuscular junction (NMJ). Previous work has shown high expression of the endocytic recycling regulator Eps15 homology domain-containing (EHD)1 protein in the Torpedo californica electric organ, a model tissue for investigating a cholinergic synapse. This study investigated the localization of EHD1 and its paralogs EHD2, EHD3, and EHD4 in mouse skeletal muscle, and assessed the morphological changes in EHD1-/- NMJs. Localization of the candidate NMJ protein EHD1 was assessed by confocal microscopy analysis of whole-mount mouse skeletal muscle fibers after direct gene transfer and immunolabeling. The potential function of EHD1 was assessed by specific force measurement and α-bungarotoxin-based endplate morphology mapping in EHD1-/- mouse skeletal muscle. Endogenous EHD1 localized to primary synaptic clefts of murine NMJ, and this localization was confirmed by expression of recombinant green fluorescent protein labeled-EHD1 in murine skeletal muscle in vivo. EHD1-/- mouse skeletal muscle had normal histology and NMJ morphology, and normal specific force generation during muscle contraction. The EHD 1-4 proteins showed differential localization in skeletal muscle: EHD2 to muscle vasculature, EHD3 to perisynaptic regions, and EHD4 to perinuclear regions and to primary synaptic clefts, but at lower levels than EHD1. Additionally, specific antibodies raised against mammalian EHD1-4 recognized proteins of the expected mass in the T. californica electric organ. Finally, EHD4 expression was more abundant in EHD1-/- mouse skeletal muscle than in wild-type skeletal muscle. It is concluded EHD1 and EHD4 localize to the primary synaptic clefts of the NMJ. Lack of obvious defects in NMJ structure and muscle function in EHD1-/- muscle may be due to functional compensation by other EHD paralogs (Mate, 2012).

Sarcolemmal repair is a slow process and includes EHD2

Skeletal muscle is continually subjected to microinjuries that must be repaired to maintain structure and function. Fluorescent dye influx after laser injury of muscle fibers is a commonly used assay to study membrane repair. This approach reveals that initial resealing only takes a few seconds. However, by this method the process of membrane repair can only be studied in part and is therefore poorly understood. This study investigated membrane repair by visualizing endogenous and GFP-tagged repair proteins after laser wounding. Membrane repair and remodeling after injury is not a quick event but requires more than 20 min. The endogenous repair protein dysferlin becomes visible at the injury site after 20 seconds but accumulates further for at least 30 min. Annexin A1 and F-actin are also enriched at the wounding area. A new participant was identified in the membrane repair process, the ATPase EHD2. EHD2, but not EHD1 or mutant EHD2, accumulates at the site of injury in human myotubes and at a peculiar structure that develops during membrane remodeling, the repair dome. In conclusion, this study established an approach to visualize membrane repair that allows a new understanding of the spatial and temporal events involved (Marg, 2012).

Regulation of synaptic vesicle budding and dynamin function by an EHD ATPase

Eps15 homology domain-containing proteins (EHDs) are conserved ATPases implicated in membrane remodeling. Recently, EHD1 was found to be enriched at synaptic release sites, suggesting a possible involvement in the trafficking of synaptic vesicles. This study investigated the role of an EHD1/3 ortholog (l-EHD) in the lamprey giant reticulospinal synapse. l-EHD was detected by immunogold at endocytic structures adjacent to release sites. In antibody microinjection experiments, perturbation of l-EHD inhibited synaptic vesicle endocytosis and caused accumulation of clathrin-coated pits with atypical, elongated necks. The necks were covered with helix-like material containing dynamin. To test whether l-EHD directly interferes with dynamin function, fluid-supported bilayers were used as in vitro assay. l-EHD was found to strongly inhibit vesicle budding induced by dynamin in the constant presence of GTP. l-EHD also inhibited dynamin-induced membrane tubulation in the presence of GTPgammaS, a phenomenon linked with dynamin helix assembly. These in vivo results demonstrate the involvement of l-EHD in clathrin/dynamin-dependent synaptic vesicle budding. Based on these in vitro observations, it is suggested that l-EHD acts to limit the formation of long, unproductive dynamin helices, thereby promoting vesicle budding (Jakobsson, 2011).

AMPH-1/Amphiphysin/Bin1 functions with RME-1/Ehd1 in endocytic recycling

RME-1/EHD1 (receptor mediated endocytosis/Eps15 homology-domain containing 1) family proteins are key residents of the recycling endosome, which are required for endosome-to-plasma membrane transport in Caenorhabditis elegans and mammals. Recent studies suggest similarities between the RME-1/EHD proteins and the Dynamin GTPase superfamily of mechanochemical pinchases, which promote membrane fission. This study showed that endogenous C. elegans AMPH-1, the only C. elegans member of the Amphiphysin/BIN1 family of BAR (Bin1-Amphiphysin-Rvs161p/167p)-domain-containing proteins, colocalizes with RME-1 on recycling endosomes in vivo, that amph-1-deletion mutants are defective in recycling endosome morphology and function, and that binding of AMPH-1 Asn-Pro-Phe(Asp/Glu) sequences to the RME-1 EH-domain promotes the recycling of transmembrane cargo. A requirement was found for human BIN1 (also known as Amphiphysin 2) in EHD1-regulated endocytic recycling. In vitro, this study found that purified recombinant AMPH-1-RME-1 complexes produce short, coated membrane tubules that are qualitatively distinct from those produced by either protein alone. These results indicate that AMPH-1 and RME-1 cooperatively regulate endocytic recycling, probably through functions required for the production of cargo carriers that exit the recycling endosome for the cell surface (Pant, 2009).


Search PubMed for articles about Drosophila Past1

Anggono, V., Koc-Schmitz, Y., Widagdo, J., Kormann, J., Quan, A., Chen, C. M., Robinson, P. J., Choi, S. Y., Linden, D. J., Plomann, M. and Huganir, R. L. (2013). PICK1 interacts with PACSIN to regulate AMPA receptor internalization and cerebellar long-term depression. Proc Natl Acad Sci U S A 110(34): 13976-13981. PubMed ID: 23918399

Braun, A., Pinyol, R., Dahlhaus, R., Koch, D., Fonarev, P., Grant, B. D., Kessels, M. M. and Qualmann, B. (2005). EHD proteins associate with syndapin I and II and such interactions play a crucial role in endosomal recycling. Mol Biol Cell 16(8): 3642-3658. PubMed ID: 15930129

Curran, J., Makara, M. A., Little, S. C., Musa, H., Liu, B., Wu, X., Polina, I., Alecusan, J. S., Wright, P., Li, J., Billman, G. E., Boyden, P. A., Gyorke, S., Band, H., Hund, T. J. and Mohler, P. J. (2014). EHD3-dependent endosome pathway regulates cardiac membrane excitability and physiology. Circ Res 115(1): 68-78. PubMed ID: 24759929

Dorot, O., Steller, H., Segal, D. and Horowitz, M. (2017). Past1 modulates Drosophila eye development. PLoS One 12(1): e0169639. PubMed ID: 28060904

Gorczyca, D., Ashley, J., Speese, S., Gherbesi, N., Thomas, U., Gundelfinger, E., Gramates, L. S. and Budnik, V. (2007). Postsynaptic membrane addition depends on the Discs-Large-interacting t-SNARE Gtaxin. J Neurosci 27(5): 1033-1044. PubMed ID: 17267557

Jakobsson, J., Ackermann, F., Andersson, F., Larhammar, D., Low, P. and Brodin, L. (2011). Regulation of synaptic vesicle budding and dynamin function by an EHD ATPase. J Neurosci 31(39): 13972-13980. PubMed ID: 21957258

Koles, K., Messelaar, E. M., Feiger, Z., Yu, C. J., Frank, C. A. and Rodal, A. A. (2015). The EHD protein Past1 controls postsynaptic membrane elaboration and synaptic function. Mol Biol Cell 26(18):3275-88. PubMed ID: 26202464

Marg, A., Schoewel, V., Timmel, T., Schulze, A., Shah, C., Daumke, O. and Spuler, S. (2012). Sarcolemmal repair is a slow process and includes EHD2. Traffic 13(9): 1286-1294. PubMed ID: 22679923

Masuda, M. and Mochizuki, N. (2010). Structural characteristics of BAR domain superfamily to sculpt the membrane. Semin Cell Dev Biol 21(4): 391-398. PubMed ID: 20083215

Mate, S. E., Van Der Meulen, J. H., Arya, P., Bhattacharyya, S., Band, H. and Hoffman, E. P. (2012). Eps homology domain endosomal transport proteins differentially localize to the neuromuscular junction. Skelet Muscle 2(1): 19. PubMed ID: 22974368

Melo, A. A., Hegde, B. G., Shah, C., Larsson, E., Isas, J. M., Kunz, S., Lundmark, R., Langen, R. and Daumke, O. (2017). Structural insights into the activation mechanism of dynamin-like EHD ATPases. Proc Natl Acad Sci U S A 114(22): 5629-5634. PubMed ID: 28228524

Naslavsky, N. and Caplan, S. (2011). EHD proteins: key conductors of endocytic transport. Trends Cell Biol 21(2): 122-131. PubMed ID: 21067929

Olswang-Kutz, Y., Gertel, Y., Benjamin, S., Sela, O., Pekar, O., Arama, E., Steller, H., Horowitz, M. and Segal, D. (2009). Drosophila Past1 is involved in endocytosis and is required for germline development and survival of the adult fly. J Cell Sci 122(Pt 4): 471-480. PubMed ID: 19174465

Pant, S., Sharma, M., Patel, K., Caplan, S., Carr, C. M. and Grant, B. D. (2009). AMPH-1/Amphiphysin/Bin1 functions with RME-1/Ehd1 in endocytic recycling. Nat Cell Biol 11(12): 1399-1410. PubMed ID: 19915558

Teodoro, R. O., Pekkurnaz, G., Nasser, A., Higashi-Kovtun, M. E., Balakireva, M., McLachlan, I. G., Camonis, J. and Schwarz, T. L. (2013). Ral mediates activity-dependent growth of postsynaptic membranes via recruitment of the exocyst. EMBO J 32(14): 2039-2055. PubMed ID: 23812009

Tomlinson, A., Mavromatakis, Y. E. and Struhl, G. (2011). Three distinct roles for notch in Drosophila R7 photoreceptor specification. PLoS Biol 9(8): e1001132. PubMed ID: 21886484

Yeow, I., Howard, G., Chadwick, J., Mendoza-Topaz, C., Hansen, C. G., Nichols, B. J. and Shvets, E. (2017). EHD proteins cooperate to generate caveolar clusters and to maintain caveolae during repeated mechanical stress. Curr Biol 27(19): 2951-2962. PubMed ID: 28943089

Xu, Y., Shi, H., Wei, S., Wong, S. H. and Hong, W. (2004). Mutually exclusive interactions of EHD1 with GS32 and syndapin II. Mol Membr Biol 21(4): 269-277. PubMed ID: 15371016

Biological Overview

date revised: 26 January 2018

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