InteractiveFly: GeneBrief

Myosin 61F: Biological Overview | References

Drosophila myosin IB (Myo1B) is one of two class I myosins in the Drosophila genome. In the larval and adult midgut enterocyte, Myo1B is present within the microvillus (MV) of the apical brush border (BB) where it forms lateral tethers between the MV membrane and underlying actin filament core. Expression of green fluorescent protein-Myo1B tail domain in the larval gut showed that the tail domain is sufficient for localization of Myo1B to the BB. A Myo1B deletion mutation exhibited normal larval gut physiology with respect to food uptake, clearance, and pH regulation. However, there is a threefold increase in terminal deoxynucleotidyl transferase dUTP nick-end labeling-positive enterocyte nuclei in the Myo1B mutant. Ultrastructural analysis of mutant midgut revealed many perturbations in the BB, including membrane tethering defects, MV vesiculation, and membrane shedding. The apical localization of both Dinged (fascin) and Moesin is impaired. BBs isolated from mutant and control midgut revealed that the loss of Myo1B causes the BB membrane and underlying cytoskeleton to become destabilized. Myo1B mutant larvae also exhibit enhanced sensitivity to oral infection by the bacterial pathogen Pseudomonas entomophila, and severe cytoskeletal defects are observed in the BB of proximal midgut epithelial cells soon after infection. Resistance to P. entomophila infection is restored in Myo1B mutant larvae expressing a Myo1B transgene. These results indicate that Myo1B may play a role in the local midgut response pathway of the Imd innate immune response to Gram-negative bacterial infection (Hegan, 2007).

These results demonstrate a role for Myo1B in maintaining the structural integrity of the BB domain in the larval midgut enterocyte and in providing resistance against oral infection by bacterial pathogens. Although Drosophila Myo1B is structurally most similar to vertebrate Myo1c, it seems to be the functional homologue of vertebrate Myo1a, because both of these myosins form lateral links that tether the MV membrane to the underlying actin core in the gut epithelium. A subset of the consequences of loss of Myo1B are reminiscent of the perturbations of the BB domain observed in the Myo1a knockout (KO) mouse (Tyska, 2005), although there are distinct differences between the two organisms. On the whole, the mild alterations in BB structure in the Myo1B mutant enterocyte, including irregular MV morphology and membrane shedding were less dramatic than that observed in the Myo1a KO mouse. For example, in the mouse KO, many microvilli have a vesiculated, sausage-link appearance resulting from severing of the MV core actin filaments by villin. This is presumably due to elevated intramicrovillar Ca2+ resulting from impaired Ca2+ buffering due to the absence of Myo1a-associated calmodulin light chains. In contrast, Quail, the Drosophila homologue of villin (note that Quail lacks filament severing and capping activity), is not expressed in midgut, although other members of the gelsolin family of actin capping and severing proteins may be expressed based on the detection of villin immunogens in the midgut of Manduca larvae. It will be important in future studies to determine whether the Myo1B-associated light chains (presumably calmodulin) play a similar role to mouse Myo1a in contributing to cytosolic Ca2+ homeostasis. For example, Ca2+ cytotoxicity could contribute to the elevated apoptosis observed in the Myo1B mutant (Hegan, 2007).

Although the perturbations of the midgut BB are relatively mild in the Myo1B mutant compared with the Myo1a KO mouse BB, Myo1B may play an even more critical role than mouse Myo1a in stabilizing the BB membrane cytoskeleton, based on the fragility of the Myo1B mutant BB in response to mechanical lysis. In contrast to the mouse Myo1a KO, few, if any normal looking BBs can be recovered from the Myo1B mutant gut. This destabilization may be due to the additive effects of the loss of actin-membrane tethering by both Myo1B and Dmoesin, together with the diminished filament bundling activity by singed. It is also possible that due to the small number of class I myosins expressed in the Drosophila enterocyte, Myo1B may have a larger repertoire of functions than Myo1a in the vertebrate enterocyte, in which at least three other Myo1s are also expressed (Myo1b, c, and e), whereas in Drosophila, Myo1A is the only other Myo1 present - a myosin that has been shown to have antagonistic functions with Myo1B in determination of left-right asymmetry. Indeed, in the mouse Myo1a KO enterocyte, myosin Ic is ectopically recruited to the BB from the basolateral domain, effecting partial rescue of the loss of lipid-raft-associated membrane proteins, including sucrase isomaltase. In contrast, Drosophila Myo1A remains in the subapical domain in the Myo1B mutant (Hegan, 2007).

The reduced apical localization of Dmoesin in the Myo1B mutant midgut is of particular interest given the critical roles Moesin plays in establishment of epithelial cell polarity. The absence of Myo1B mechanochemical linkages to the membrane could have an indirect impact on Moesin-membrane interactions due to altered biophysical and/or compositional changes in the BB membrane. Conversely, there may be direct interactions between these two membrane-actin linker proteins. One important question for future studies will be to determine whether loss of Myo1B results in reduced levels of active, phosphorylated Moesin (Hegan, 2007).

In contrast to Myo1B and Moesin, Singed does not exhibit tight localization to the BB domain in most regions of the gut. Nevertheless, Singed could play a key role in MV actin bundle structure, a role that could be modulated by Myo1B actin-membrane interactions. Singed is required for actin bundle assembly in nurse cells during oogenesis, yet Singed exhibits diffuse cytoplasmic localization rather than bundle association characteristic of other components such as Quail that also contribute to bundle formation. Moreover, the loss of apical localization in epithelial cells of the proventriculus and diminishment of association with invaginations of the cuprophilic cells in the Myo1B mutant indicate that Myo1B motor linkages between filaments and the membrane can also contribute to the localization/recruitment of Singed to actin bundles (Hegan, 2007).

In contrast to the vertebrate BB membrane, the physiological roles of the enterocyte in nutrient absorption and digestion in the Drosophila midgut are not well understood at the molecular level. However, the presumed absorptive and secretory functions required for lumenal nutrient digestion and nutrient uptake are not discernibly affected in the absence of Myo1B given that larval growth is not severely impaired and the complex regulation of lumenal pH along the length of the midgut is normal. However, loss of Myo1B does cause a threefold increase in the number of apoptotic enterocytes, suggesting that there is an elevated level of stress in these cells. Similar results were seen in the Myo1a KO mouse. Thus, although both Myo1B mutant larvae and Myo1a KO mice exhibit normal growth rates, indicative of adequate nutrient absorption under ideal laboratory feeding and living environments, the increased cellular stress in these mutants may have significant consequences under less than ideal and more realistic environments (Hegan, 2007).

In vertebrates, the intestinal mucosa plays essential roles in the barrier functions of the gut in response to mucosal injury, inflammation, and to pathogens. The role of the Drosophila midgut epithelium in providing a similar barrier is less clear, but recent work has highlighted the importance of the Drosophila midgut epithelium during the innate immune response. In contrast to the vertebrate gut, the lumen of the fly midgut is lined by a tube of extracellular matrix, the peritrophic membrane, which serves as a physical barrier between the epithelium and potential fungal and bacterial pathogens. However, this barrier is permeable to digested nutrients and presumably any toxins released by ingested pathogens. Indeed, the recent studies of Lamaitre and colleagues (Vodovar, 2005; Liehl, 2006) on the response to oral infection by the lethal bacterial pathogen P. entomophila have provided convincing evidence for a localized response by the midgut epithelium in addition to the systemic innate immune response after pathogen infection (Hegan, 2007).

P. entomophila is one of the few Drosophila pathogens identified that is lethal after oral infection (Vodovar, 2005). Oral infection of P. entomophila elicits a local immune response in the gut that includes secretion of the antibacterial peptide diptericin as well activation of the systemic Imd immune response pathway (Liehl, 2006). One of the consequences of P. entomophila infection is disruption of the BB domain at relatively late stages of infection, followed by an increase in Myo1B gene expression (Vodovar, 2005), suggesting that the midgut epithelium may play an important role in host resistance to infection and the resultant response to toxins released by P. entomophila into the gut lumen. This possibility is underscored by the observations reported in this study that Myo1B mutant larvae are hypersensitive to P. entomophila infection, and these larvae exhibit more rapid disruptions of the midgut epithelium after oral infection with P. entomophila, compared with Myo1B heterozygous larvae (Hegan, 2007).

The molecular basis for the role of Myo1B in conferring resistance to infection by P. entomophila is not known. The simplest possibility is that destabilization of the BB domain by loss of Myo1B membrane tethering makes the BB membrane more susceptible to damage by P. entomophila secreted toxins (e.g., lipases; Vodovar, 2006). There may also be compositional changes in the BB membrane if Myo1B is involved in the transport to and/or retention of proteins in the BB membrane, as has been demonstrated for retention of lipid raft proteins by vertebrate Myo1a (Tyska, 2004; Tyska, 2005). Another possibility is that Myo1B plays an active role in secretion of antibacterial peptides such as diptericin. In this regard, it will be of interest to see whether the Myo1B mutants show increased sensitivity to the secreted P. entomophila metalloprotease, AprA (whose presumed targets may include diptericin), which in the absence of bacteria is sufficient to elicit the local gut immune response after oral ingestion (Liehl, 2006). Although the basis for the role of Myo1B in conferring pathogen resistance is unknown, the results presented in this study demonstrate a novel role for this class I myosin in the barrier functions of the midgut epithelium that is quite distinct from its role, together with Myo1A, in determining right left visceral symmetry (Hegan, 2007).

An unconventional myosin in Drosophila reverses the default handedness in visceral organs

The internal organs of animals often have left-right asymmetry. Although the formation of the anterior-posterior and dorsal-ventral axes in Drosophila is well understood, left-right asymmetry has not been extensively studied. This study demonsrates that the handedness of the embryonic gut and the adult gut and testes is reversed (not randomized) in viable and fertile homozygous Myo31DF mutants. Myo31DF encodes an unconventional myosin, Drosophila MyoIA [also referred to as MyoID in mammals; Gillespie, 2001; Morgan, 1995), and is the first actin-based motor protein to be implicated in left-right patterning. Myo31DF is required in the hindgut epithelium for normal embryonic handedness. Disruption of actin filaments in the hindgut epithelium randomizes the handedness of the embryonic gut, suggesting that Myo31DF function requires the actin cytoskeleton. Consistent with this, it was found that Myo31DF colocalizes with the cytoskeleton. Overexpression of Myo61F (myosin-IB), another myosin I, reverses the handedness of the embryonic gut, and its knockdown also causes a left-right patterning defect. These two unconventional myosin I proteins may have antagonistic functions in left-right patterning. It is suggested that the actin cytoskeleton and myosin I proteins may be crucial for generating left-right asymmetry in invertebrates (Hozumi, 2006). An accompanying paper (Spéder, 2006) demonstrates that Myo31DF regulates dextral looping of genitalia.

Mechanisms that create characteristic left-right asymmetry have been studied extensively in vertebrates. However, although the organs of many invertebrate species also have left-right asymmetry, the mechanisms by which this asymmetry arises are largely unknown. In Drosophila, several organs have left-right asymmetry, including the embryonic gut, the adult brain and the genitalia (Hozumi, 2006).

To identify genes involved in left-right asymmetry of the Drosophila embryonic gut, a genetic screen was performed using a collection of P-element lines. The embryonic gut is composed of three major parts, the foregut, midgut and hindgut, all of which have characteristic left-right asymmetry. 75.7% of homozygous Myo31DF^souther embryos show synchronous inversion of the midgut and hindgut. In these embryos, the hindgut and midgut are the mirror-image of those in wild-type embryos, rather than showing randomized patterning. In contrast, foregut handedness was normal in all cases examined, indicating that this phenotype was heterotaxic. Myo31DF^souther is a background mutation of the Gene Search Drosophila line GS14508. Deficiency mapping was used to map the cytological location of Myo31DF^souther to between 30D and 31F. Complementation tests were performed between Myo31DF^souther and lines bearing mutations that map to this region. Myo31DF^souther failed to complement Myo31DF^K1 (Spéder, 2006), an allele of Myo31DF encoding Drosophila MyoID (Hozumi, 2006).

The transposable element gypsy was inserted into the 5'-untranslated region of the Myo31DF gene in Myo31DF^souther. Myo31DF^L152 was one of five ethylmethanesulfonate (EMS)-induced Myo31DF alleles isolated in a large-scale EMS mutant screen. Myo31DF^L152 has a base substitution that introduces a premature stop codon at amino acid 331, resulting in a putative truncated product. Myo31DF overexpression from UAS-Myo31DF driven by byn-Gal4 in the hindgut and posterior midgut and their primordial counterparts rescued the left-right defects of Myo31DF^souther embryos, indicating that Myo31DF was responsible for the heterotaxia. In Myo31DF^souther embryos, NP2432-driven expression of Myo31DF in the hindgut epithelium, but not in other parts of the embryonic gut, such as the midgut and mesoderm, was sufficient to rescue this heterotaxia, suggesting that Myo31DF is required in the hindgut epithelium. Furthermore, the frequency of the handedness defect was similar in Myo31DF^L152 homozygous and Myo31DF^L152/Df(2L)J2 embryos. Thus, Myo31DF^L152 is probably a null mutant of Myo31DF. All homozygous Myo31DF mutants isolated in this study were viable and fertile, with normal hindgut tissue specification, suggesting that Myo31DF function is largely restricted to left-right patterning. No maternal phenotype or Myo31DF gene contribution was detected. Notably, the foregut became a mirror-image of its wild-type counterpart when Myo31DF was overexpressed in the entire embryo, but other parts of the gut were normal. Therefore, it is suggested that Myo31DF is not involved in the left-right asymmetrical development of the foregut in wild-type embryos, but can reverse foregut handedness (Hozumi, 2006).

The adult hindgut and testes, which are regenerated during metamorphosis, were examined. These also showed inversed handedness in the Myo31DF homozygote. In most Myo31DF^L152 adults, the loop of the hindgut and spiral of the testes were reversed, although not always synchronously. The function of the Myo31DF gene was knocked-down using RNA interference (RNAi) in vivo. The expression of double-stranded Myo31DF RNA driven by byn-Gal4 caused inversion of the adult, but not the embryonic, hindgut. Thus, the left-right pattern involving Myo31DF is not transmitted during metamorphosis (Hozumi, 2006).

In situ hybridization revealed Myo31DF expression in the amnioproctodeal invagination at stage 6. At stages 12-14, Myo31DF messenger RNA was strongly detected in the primordial midgut and hindgut, and in the proventriculus, midgut and hindgut. A sense-strand probe of Myo31DF gave no detectable signal. Immunostaining of wild-type embryos with an anti-Myo31DF antibody (anti-Myo31DF-1P) also labelled the midgut and hindgut. These signals were absent in Myo31DF^souther and Myo31DF^L152 homozygotes, indicating that the staining was specific. Myo31DF mRNA and protein were detected in a symmetrical pattern before the development of left-right asymmetry. Myo31DF protein is expressed in the adult gut (Morgan, 1995). No Myo31DF expression was detected in the foregut, which may account for the absence of any laterality defect in the foregut of Myo31DF mutants (Hozumi, 2006).

Myo31DF protein binds to actin in an ATP-dependent manner (Morgan, 1995). The co-localization of Myo31DF was examined and the actin cytoskeleton in cultured Drosophila S2 cells. A green fluorescent protein (GFP)-tagged Myo31DF (Myo31DF-GFP) had wild-type function, given that its overexpression rescued Myo31DF^souther. Myo31DF-GFP co-localized with actin, mostly at cell protrusions. In epithelial cells of the hindgut, endogenous Myo31DF was detected as punctate staining, partly overlapping with cortical actin (Hozumi, 2006).

byn-Gal4-driven misexpression of GFP-tagged moesin, an actin-binding protein, in wild-type embryos caused a reduction in actin filaments in the apical region of the hindgut epithelium, where Myo31DF function is required. Notably, the midgut and hindgut always had the same handedness, but the handedness was random (not inversed), in wild-type and Myo31DF homozygous embryos. Embryo handedness was also affected by NP2432-driven GFP-moesin expressed in the hindgut epithelium only. However, GFP-moesin expression in the midgut only did not affect handedness (Hozumi, 2006).

To investigate further the functional link between Myo31DF and the actin cytoskeleton, the phenocritical period for inducing left-right defects was determined using Myo31DF RNAi and GFP-moesin misexpression, following the TARGET method. Both Myo31DF knockdown and GFP-moesin expression in the hindgut 0-24 h after pupation caused similar defects in adult gut handedness, suggesting that Myo31DF and the appropriate organization of actin filaments are required at the same time. GFP-moesin also affected handedness in the Myo31DF embryo, suggesting that the default handedness, which may be manifested in the Myo31DF homozygote, also depends on the actin cytoskeleton. The involvement of three Rho GTPase family proteins, Rho, Rac and Cdc42, which regulate the organization of the actin cytoskeleton, was examined. Expression of dominant-negative forms of these proteins, especially Rho, in the hindgut induced synchronous left-right defects in the embryonic midgut and hindgut. Together, these results suggest that Myo31DF depends on the actin cytoskeleton to generate left-right asymmetry (Hozumi, 2006).

Cell division and cell death do not occur during left-right asymmetric development of the hindgut. It is therefore speculated that the rearrangement of hindgut epithelial cells may be part of this process. To test this possibility, a time-lapse analysis was performed. The position of each cell was visualized by labelling the nucleus with GFP. Cell rearrangement, which coincided with the left-right bias associated with left-handed rotation of the hindgut, was suggested by the significant intercalation of some cells (Hozumi, 2006).

Another myosin I protein, Myo61F (Drosophila MyoIB, also referred to as MyoIC in mammals), has been reported in Drosophila. Myo61F protein is detected in the embryo and adult gut. To test whether Myo61F is also involved in left-right asymmetry, Myo61F was overexpressed using UAS-Myo61F or GS9889 driven by byn-Gal4. Unexpectedly, Myo61F overexpression resulted in inversion of the midgut and hindgut in both cases. In contrast, Myo31DF overexpression did not affect the handedness of these organs. These results suggest that Myo31DF and Myo61F have antagonistic functions in creating the left-right asymmetry of these organs. The involvement of Myo61F in left-right asymmetry is also supported by the finding that its knockdown by RNAi results in the left-right defect in the embryonic midgut (Hozumi, 2006).

Homozygous Myo31DF embryos show reversed handedness of embryonic and adult visceral organs, which may represent the default state of left-right asymmetry in Drosophila. This situation is similar to the function of the sinistral gene in the freshwater snail, Limnea (although the sinistral gene is required maternally). Normal handedness is still seen in 25% of Myo31DF homozygotes. It is speculate that some other myosin gene(s) has a redundant function in left-right patterning. Inversion of the anteroposterior axis does not affect laterality, suggesting that left-right pattering occurs zygotically; this is consistent with the zygotic function of Myo31DF. These results also suggest that an actin-based mechanism, which can align itself to either an anteroposterior-dorsoventral reference or the pre-existing sinistral handedness, exists to direct the rotation of the hindgut epithelium. Since myosin I proteins are involved in vesicular transport, it is proposed that Myo31DF and Myo61F, which on the basis of their structures are believed to move to the plus ends of actin filaments, carry left-right determinants with opposite activities. Thus, both left-right determinants would be concentrated in the plus ends of actin filaments that have a hypothetical planar polarity. In the Myo31DF mutant, only the opposing determinant is concentrated here, which reverses the handedness. According to this model, disruption in actin organization would result in left-right randomization, as was indeed observed experimentally (Hozumi, 2006).

Myosin-1a powers the sliding of apical membrane along microvillar actin bundles

Microvilli are actin-rich membrane protrusions common to a variety of epithelial cell types. Within microvilli of the mammalian enterocyte brush border (BB), myosin-1a (Myo1a) forms an ordered ensemble of bridges that link the plasma membrane to the underlying polarized actin bundle. Despite decades of investigation, the function of this unique actomyosin array has remained unclear. This study show that addition of ATP to isolated BBs induces a plus end-directed translation of apical membrane along microvillar actin bundles. Upon reaching microvillar tips, membrane is 'shed' into solution in the form of small vesicles. Because this movement demonstrates the polarity, velocity, and nucleotide dependence expected for a Myo1a-driven process, and BBs lacking Myo1a fail to undergo membrane translation, it is concluded that Myo1a powers this novel form of motility. Thus, in addition to providing a means for amplifying apical surface area, it is proposed that microvilli function as actomyosin contractile arrays that power the release of BB membrane vesicles into the intestinal lumen (McConnell, 2007).

This paper describes a novel form of microvillar contractility that can be reactivated by exposing native, isolated BBs to ATP. Reactivation is manifest as the plus end-directed movement of apical membrane along microvillar core actin bundles, and eventually, the accumulation of membrane at microvillar tips. Upon reaching the tips, the membrane no longer maintains contact with the underlying actin cytoskeleton and vesiculation is favored; small vesicles that contain Myo1a and are enriched in other apical membrane markers are released into solution. The translation and shedding of membrane requires ATP hydrolysis, demonstrates the nucleotide dependence expected for a myosin ATPase, and is substantially depressed in the absence of Myo1a. Intriguingly, the membrane translation velocities are nearly identical to velocities measured in sliding filament assays with Myo1a immobilized on lipid-coated coverslips. Myo1a is a good candidate for driving this novel form of motility, as previous studies have established that this motor can bind directly to apical membrane lipids and proteins, is present at a very high concentration (>70 microM) in the microvillus, and moves toward the plus-ends of actin filaments in vitro. Thus, in combination with previous studies, these data strongly indicate that Myo1a is the motor that powers ATP-stimulated membrane translation and shedding in isolated BBs (McConnell, 2007).

These findings demonstrate that the polarized actin bundles that support microvilli serve as tracks for myosin-based motor activity. Although these studies focus on Myo1a, a reasonable extension of this is that other myosins in the BB may also use core actin bundles as tracks for directed transport. Consistent with this idea, recent studies in the context of kidney proximal tubule reveal that hypertension induces the redistribution of Myo6 immunoreactivity along the microvillar axis. The data also show that microvillar Myo1a is mechanochemically active and generates force in its native environment. This suggests that Myo1a may function as more than a passive 'linker' serving to stabilize membrane/cytoskeleton interactions (Tyska, 2005). Finally, while general models for myosin-I function have always included some form of mechanical activity involving membrane rearrangement or movement, direct evidence for these functions has been lacking. The data presented in this study demonstrate directly, in a native system, that Myo1a is capable of producing mechanical forces sufficient to power the movement of cellular membranes over the actin cytoskeleton (McConnell, 2007).

A number of previous biochemical studies have documented the existence of small vesicles in the lumen of the small intestine. Although these vesicles are enriched in nutrient-processing enzymes in a manner similar to BB membrane, their mechanism of release remains unclear. Vesiculation from the tips of microvilli has also been captured in ultrastructural studies of intact enterocytes, suggesting that lumenal vesicles may originate from microvillar membrane. It is proposed that the Myo1a-dependent mechanical activity described in this study provides a mechanism for the formation and release of vesicles from enterocyte BBs in vivo. This model becomes even more appealing if it is considered that membrane shedding from microvilli in vivo is accelerated when enterocytes are treated with antibodies against sucrase isomaltase (SI), a transmembrane disaccharidase that interacts directly with Myo1a in a raft-like complex in the microvillar membrane (Tyska, 2004). The shedding of vesicles from the plasma membrane is a well-documented activity performed by a variety of epithelial cell types under normal and pathological conditions. In the gastrointestinal tract, the release of membrane vesicles laden with nutrient-processing enzymes could serve to increase the effective apical membrane surface area; such vesicles would allow processing to begin before nutrients reached the actual surface of the enterocyte. It has also been proposed that BB membrane shedding may allow the enterocyte to continually modify its apical membrane composition. This form of plasticity may be a critical aspect of the enterocyte response to the shifting demands in nutrient processing and absorption that are commonplace in the small intestine (McConnell, 2007).

Does the membrane shedding process described in this study represent a general function for microvilli found on other polarized cell types? Although the expression of Myo1a is restricted to the gastrointestinal tract and inner ear, other closely related class I myosins (Myo1b, Myo1c, and Myo1d) are more widely expressed in polarized cells from a variety of tissues (including kidney, liver, and pancreas) and in some cases are known to localize to microvilli. Interestingly, previous studies have established that all of these tissues release vesicles into their lumens. Thus, further studies are required to determine whether the activity described in this paper represents a general function for class I myosins expressed in other cell types, or a phenomenon specific to the gastrointestinal tract (McConnell, 2007).

In recent studies with the Myo1a KO mouse, herniations were observed of the apical domain, where large regions of BB membrane were detached from underlying microvillar actin bundles (Tyska, 2005). It was originally proposed that these herniations arise due to a lack of membrane/cytoskeleton adhesion normally provided by Myo1a. However, the new findings suggest an alternative explanation: herniations may represent excess apical membrane in the BB. In KO enterocytes, the apical sorting machinery continues to deliver apical domain components to the BB. However, in the absence of Myo1a and its associated plus end-directed mechanical activity, membrane release from microvillar tips may be slowed, resulting in the accumulation of membrane in the BB, and ultimately the formation of membrane herniations (McConnell, 2007).

Early ultrastructural studies of the enterocyte BB revealed the presence of a prominent electron-dense plaque at the distal tips of microvilli. To date, the role of the tip complex remains unclear, but by analogy to similar structures in stereocilia and filopodia, it is suspected that at least one function may be in controlling the dimensions and dynamics of the core actin bundle. Does the tip complex also play a role in the process of membrane vesiculation from microvillar tips as described in this paper? It remains possible that an additional role for this complex may be in the formation of vesicles, perhaps through promoting and/or stabilizing the high curvature of membrane that envelops the microvillus tip. This would be analogous to the activity of viral proteins such as VSV-M, which are known to be involved in curving membrane through direct interactions with lipids. Alternatively, this complex may contain proteins that actively promote fission and vesicle release. In either case, the tip complex must be dynamically reassembled after vesicle release or somehow left behind during the process. The former idea finds support in recent proteomic studies, which show that one of the few proteins known to localize to microvillar tips, Eps8, is found in vesicles released from the apical surface of cultured intestinal epithelial cells (McConnell, 2007).

Early experiments with isolated BBs established that ATP induced a contraction of the junctional band of actin filaments surrounding the terminal web region. A number of groups actively investigated this contractility and the involvement of Myo2, which at the time was the only known force generator in the BB. The findings presented in this study demonstrate that terminal web contraction and microvillar membrane translation/shedding are independent and separable activities. Thus, it is proposed that the BB contains two distinct actomyosin contractile arrays: (1) a Myo2-based array that powers the contraction of the junctional band surrounding the terminal web region, and (2) a Myo1a-based array in microvilli that exerts plus end-directed forces on the apical membrane (McConnell, 2007).

In summary, the classic view of microvillar function suggests these structures serve to enhance the efficiency of nutrient uptake by amplifying the apical membrane surface area available for nutrient processing and absorption. The work presented in this study suggests that microvilli also function as actomyosin contractile arrays, allowing for the plus end-directed movement and shedding of BB membrane from microvillar tips. Because this process may have considerable implications with regard to gastrointestinal physiology, future studies will focus on investigating how Myo1a contributes to BB membrane shedding in vivo (McConnell, 2007).

Nucleus to synapse Nesprin1 railroad tracks direct synapse maturation through RNA localization

An important mechanism underlying synapse development and plasticity is the localization of mRNAs that travel from the nucleus to synaptic sites. This study demonstrates that the giant nuclear-associated Nesprin1 (dNesp1 - FlyBase name Muscle-specific protein 300 kDa) forms striated F-actin-based filaments, which were dubbed "railroad tracks," that span from muscle nuclei to postsynaptic sites at the neuromuscular junction in Drosophila. These railroad tracks specifically wrap around immature boutons formed during development and in response to electrical activity. In the absence of dNesp1, mRNAs normally localized at postsynaptic sites are lacking and synaptic maturation is inhibited. This dNesp1 function does not depend on direct association of dNesp1 isoforms with the nuclear envelope. It was also show that dNesp1 functions with an unconventional myosin, Myo1D, and that both dNesp1 and Myo1D are mutually required for their localization to immature boutons. These studies unravel a novel pathway directing the transport of mRNAs from the nucleus to postsynaptic sites during synaptic maturation (Packard, 2015).

A crucial property of synaptic connections is their ability to change, which is thought to be at the core of adaptive processes, such as learning and memory and the refinement of connectivity. A key feature of long-term changes in synaptic structure and function is the requirement for new protein synthesis. In hippocampal neurons, ribonucleoprotein (RNP) granules are transported to the base of dendritic spines, and following plasticity-eliciting stimuli, result in RNP translocation to activated spines and induction of protein synthesis (Packard, 2015).

An important, yet poorly understood question is: How are RNPs directed to their precise destinations once they exit the nucleus? Studies in several systems provide evidence for directed trafficking of RNPs by binding to kinesin and dynein motors, thus supporting a role for microtubules in this process. However, studies also implicate actin filaments or actin-based motors, such as MyosinV/Didium, in the translocation of RNPs to dendritic spines or the posterior pole of the Drosophila oocyte. In the oocyte, the precise posterior localization of oskar mRNA, required to establish the anterior-posterior axis, requires both the activities of microtubules and actin-based motors. In this process MyosinV/Didium interacts with Kinesin heavy chain, suggesting an interplay between the actin and microtubule cytoskeleton. It is proposed that microtubules could mediate long-range movements of RNPs from the nucleus to the periphery, but that precise localization of RNPs requires short-range interactions between RNPs and the actin-based cytoskeleton. However, these long versus short-range interactions are still ill defined (Packard, 2015).

To determine a potential role of the actin cytoskeleton in the postsynaptic localization of RNPs, this study focused on the actin-binding protein MSP300/Drosophila Nesprin-1 (dNesp1; also known as Syne1), a component of the LInker of Nucleoskeleton and Cytoskeleton (LINC) complex. The LINC complex links the nuclear cytoskeleton with the actin-based cytoplasmic cytoskeleton. dNesp1 is a giant transmembrane protein of the spectrin superfamily, which is associated with a variety of musculoskeletal disorders, such as X-linked Emery-Dreifuss muscular dystrophy (EDMD), movement disorders such as autosomal recessive cerebellar ataxia type 1 (ARCA1), bipolar disorder, and it is a risk gene for schizophrenia and autism. The largest isoform(s) of dNesp1 is embedded in the outer nuclear membrane (ONM) via its transmembrane domain. The C-terminal tail, containing a Klarsicht/Anc1/Syne Homology (KASH) domain, faces the nuclear intermembrane space (also referred as to the perinuclear space) between the ONM and the inner nuclear membrane (INM) and interacts with the INM Sad1/Unc84 (SUN) domain-containing proteins, thus connecting ONM and INM proteins. Its giant N-terminal domain faces the cytoplasm and contains multiple spectrin-type repeats as well as two calponin actin-binding domains. However, other dNesp1 isoforms lack the KASH domain and thus likely are not directly linked to the nuclear envelope (Packard, 2015).

At the mammalian neuromuscular junction (NMJ) Nesp1, is involved in interactions with the acetylcholine receptor (AChR) clustering molecule muscle-specific kinase (MuSK). In the central nervous system CPG2, an isoform of Syne1, participates in the trafficking of glutamate receptors (GluRs). Studies in Drosophila and mice show that Nesp1 is required for normal nuclear localization in muscle cells and the integrity of muscle cell insertion sites into the cuticle. Recently, reports suggest that dNesp1 isoforms lacking the KASH domain are also required for normal Drosophila larval locomotion, selective localization of GluRIIA and synaptic function at the NMJ, independent of its nuclear localization role. However, its potential involvement in the localization of synaptic mRNAs has not been investigated (Packard, 2015).

This study reports that interfering with dNesp1 isoforms at the Drosophila NMJ disrupts the postsynaptic localization of mRNAs in muscle, and thus the localization of the proteins encoded by these mRNAs at the postsynaptic region. In addition, mutations in dnesp1 alter synapse development and activity-dependent plasticity. In these mutants, mRNAs accumulate in the cytoplasm at the nuclear periphery, suggesting that the defect likely originates from abnormal transport of these mRNAs to synaptic sites and not from the nuclear export of these mRNAs. Strikingly in wild-type muscles, dNesp1 protein is organized into long striated filaments, dubbed 'railroad tracks,' which extend all the way from the nucleus to the periphery of the NMJ. dNesp1 railroad tracks are the first postsynaptic elements found to associate specifically with immature synaptic boutons formed during NMJ expansion or upon spaced stimulation. This study showed that dNesp1 binds to a synaptically localized RNA. In addition, dNesp1 colocalizes and cosediments with F-actin, confirming its relationship with the actin cytoskeleton. Furthermore, its exclusive localization around nascent synaptic boutons is similar to the distribution of the unconventional actin motor, Myo31DF, the Drosophila ortholog of human Myo1D. Null mutations in myo31DF mimic the phenotypes of the severe hypomorphic dnesp1^sZ75 mutant, and both dNesp1 and Myo31DF are required for each other's localization. These studies unravel a novel filamentous network connecting the nucleus to nascent synaptic boutons, and this network functions with actin motors for proper localization of postsynaptic RNPs (Packard, 2015).

mRNA localization and local translation are critical for the formation and plasticity of synaptic connections. However, the exact mechanisms involved in precisely localizing mRNAs are still unclear. This study provides evidence for a novel mechanism of mRNA delivery at the Drosophila larval NMJ, from the muscle nucleus to developing postsynaptic sites. F-actin-associated dNesp1 railroad tracks, which run through the muscle cell cortex, bridge the distance from the nuclear envelope to the NMJ. At the NMJ, these railroad tracks enwrap immature synaptic boutons becoming the first identified proteins localized to boutons, which until this point lack postsynaptic proteins. Thus, dNesp1 railroad tracks provide a pathway of communication between the nucleus and sites of synapse formation. The results suggest that dNesp1 railroad tracks serve to transport mRNAs required to build the postsynaptic machinery because severe reduction in dNesp1 results in accumulation of postsynaptically enriched transcripts at the nuclear periphery and their depletion from the NMJ. Consistent with the association of dNesp1 railroad tracks with F-actin suggested by labeling body wall muscles and by the finding that dNesp1 cosediments with F-actin, it was found that a myosin1 motor, Myo31DF, colocalizes with dNesp1. Absence of Myo31DF mimicked the synaptic phenotypes of dnesp1^sZ75 mutants. In addition, Myo31DF is required for normal association of dNesp1 with immature boutons and with transport of postsynaptic transcripts. Taken together, it is proposed that dNesp1 railroad tracks form a pathway for the polarized transport of mRNAs to immature synapses during development of postsynaptic structures. Furthermore, based on the known properties of the Myosin1 family, it is proposed that this motor is required to either anchor dNesp1 railroad tracks to the membrane in their pathway to ghost boutons, to locally polymerize actin, or serve as a motor to specifically transport RNPs to maturing postsynaptic sites (Packard, 2015).

dNesp1 filaments can go all the way from the nuclear envelope to sites of postsynaptic maturation. dNesp1 is part of the LINC complex linking the nucleoskeleton to the cytoskeleton. However, many dNesp1 isoforms lack the transmembrane and KASH domain. Whether these isoforms are still linked to the nuclear envelope through dimerization with transmembrane and KASH domain-containing isoforms is not known, but there is evidence that Nesprins can associate with each other and form filaments as observed in other proteins of the spectrin family. Particularly prominent is the giant cytoplasmically localized N-terminal rod domain of about 300-500 nm, which projects into the cytoplasm. The long rod domain contains multiple spectrin repeats similar to other proteins of this family, such as spectrin, α-actinin, dystrophin, and utrophin. Of these, α-actinin has been shown to form F-actin-based striated filaments with staggered F-actin and α-actinin striations at a similar periodicity (0.5 μm) to those described in this study for F-actin and dNesp1. If dNesp1 does behave as an antiparallel dimer, as observed with %alpha-actinin and suggested in vitro for dNesp1, the actin-binding CH domains located at each end of the dimer could bind to F-actin, in a repeated manner, forming striations. Similar striated filaments have been observed in the case of actomyosin filaments (containing MyosinII) in several cell types and believed to convey elastic properties to the cells. The current studies were unable to determine if F-actin also formed these arrangements with Myo1 because the fixation conditions to examine both proteins with antibodies and fungal toxins were incompatible. This study demonstrates that these dNesp1 striated filaments can extend all the way from the nuclear envelope to sites of postsynaptic maturation, and enwrap these sites (Packard, 2015).

These studies demonstrate a specific association between dNesp1 railroad tracks and ghost boutons that are naturally occurring in wild-type NMJs, as well as those induced by patterned electrical stimulation. Ghost boutons are thought to represent a transient state of synaptic bouton maturation in which postsynaptic proteins have not yet been recruited. So far, dNesp1 and Myo31DF are the first proteins found to be localized at the postsynaptic region of ghost boutons. This is consistent with the model that these proteins participate in the earliest events during postsynaptic maturation, particularly the localization of specific postsynaptic mRNAs. Mutations that disrupt the maturation of ghost boutons result in NMJ arbors with fewer synaptic boutons and an overall accumulation of ghost boutons. Most of these mutations are associated with alterations in Wnt signaling, which is essential for postsynaptic maturation. Interestingly, mutations in the Caenorhabditis elegans Nesprin 1, ANC-1, also led to defects in synapse formation through interaction with Wnt signaling molecules (Packard, 2015 and references therein).

In mammals, the first Nesprin 1 isoform (Syne1) was isolated in a yeast two-hybrid screen using the MuSK as bait. MuSK is a protein required for postsynaptic differentiation. Interestingly, Syne1 was found to be exclusively associated with synaptic muscle nuclei, the subset of nuclei that transcribe synaptic genes needed for postsynaptic assembly. Subsequent studies at mammalian central glutamatergic synapses revealed that CPG2, an activity-dependent brain-specific isoform of Syne-1 was present at the postsynaptic region of excitatory synapses. Altering CPG2 levels resulted in abnormal dendritic spine size and disrupted constitutive endocytosis of AMPA receptors, which is linked to synaptic plasticity. Notably, mutations in syne-1 have been linked to autosomal recessive cerebellar ataxia, Emery Dreifuss muscular dystrophy, autism, and bipolar disorder, suggesting its importance in nervous system function (Packard, 2015).

At the Drosophila NMJ, dNesp1 is also involved in regulating the subunit composition of glutamate receptors (GluRs), synaptic transmission, and larval locomotion. However, in these studies, the authors used a single dNesp1 mutation lacking the KASH domain. The current studies revealed that the KASH domain is not required for the regulation of bouton number or the localization of Par6 protein. Thus, the GluR phenotypes are most likely to represent a different function of dNesp1 in later stages of synaptic bouton maturation (Packard, 2015).

Myo31DF is a conserved protein belonging to the Myosin ID family of unconventional myosins. Class 1 myosins are monomeric and can interact with membranes through their C-terminal Tail Homology 1 (TH1) domain containing a Pleckstrin Homology (PH) lipid-binding domain. In addition, they bind to actin through their N-terminal ATPase motor head. Connecting the C- and N-terminal domains is the neck region, which binds to Calmodulin and behaves as a lever arm for force generation and membrane deformation. The monomeric nature of Myo1D makes it unlikely to function as a processive motor for cargo transport. However, MyoI ensembles have been shown to generate directed membrane movements when anchored to actin filaments. In rats, Myo1D is believed to mediate vesicular transport and fly Myo31DF interacts with dynamin. Studies in the fly have also suggested that Myo1D regulates contacts between cells because mutations in myo31DF lead to defective left-right asymmetry, a process highly dependent on adherens junctions. In the mammalian nervous system, Myo1D is found in dendrites and axons during development. As in the case of Nesprin 1, human Myo1D has also been linked to autism (Packard, 2015).

Similar to dNesp1, this study found that Myo31DF was enriched at ghost boutons, was required for activity-dependent ghost bouton formation and maturation, and was needed for proper localization of par6 and magi mRNA at the postsynaptic region of the NMJ. The remarkable similarity between the phenotypes, as well as the colocalization of the proteins at ghost boutons suggest that Nesp1 and Myo31DF function in the same early process of bouton maturation. Supporting this conclusion is the observation that dNesp1 and Myo31DF were required for each other's localization at ghost boutons and that both genes genetically interact. In myo31df mutants, cytoplasmic dNesp1 filaments were still observed, but they no longer associated with ghost boutons. Considering the properties of members of the myosin I family, it is possible that Myo31DF serves to direct and anchor dNesp1 railroad tracks to the postsynaptic membrane apposed to newly formed ghost boutons. Alternatively, or in addition, Myo31DF might be required for F-actin polymerization and thus the formation of dNesp1 railroad tracks around newly formed ghost boutons. Interestingly, Myo31DF binds to Calmodulin light chains and dNesp1 contains Calmodulin-binding sites, which might serve as a site for direct interaction (Packard, 2015).

Recently studies have determined that par6 and magi mRNAs exit the nucleus as part of large RNPs that exit the nucleus through a mechanism of budding at the nuclear envelope. Two lines of evidence suggest that the phenotypes observed in this study are unlikely to result from blocking nuclear envelope budding. First, the dnesp1^{Δ KASH} mutation, lacking the C-terminal region required to associate dNesp1 with the nuclear envelope, had normal Par6 protein levels at the NMJ and did not display the morphological NMJ defects associated with the severe hypomorphic dnesp1^sZ75 mutant. Second, in dnesp1^sZ75 mutants par6 and magi RNAs were observed in the cytoplasm, suggesting that they are exported from the nucleus. However, they accumulated around the nucleus and were not transported to postsynaptic sites. It is proposed that in the absence of dNesp1 railroad tracks in the severe hypomorphic dnesp1^sZ75 mutant, megaRNPs fail to be transported in a polarized manner to the postsynaptic region of the NMJ (Packard, 2015).

In some systems, such as the Drosophila embryo, RNA localization appears to be a major mechanism for the regulation of translation. The localization of mRNAs at postsynaptic sites allows a rapid and synapse-specific translation of plasticity related transcripts in response to appropriate patterns of electrical activity, which appear essential for long-term synaptic plasticity. Studies of RNA localization to synapses and other cellular regions have implicated both microtubules and kinesin motors, as well as F-actin and myosin motors, in transporting RNPs to their site of translation. It has been suggested that microtubules constitute a long-range transport mechanism for RNP transport to sites close to the membrane whereas microfilaments may serve as a short-range transporters at the cellular cortex, with the unconventional myosins V and VI and the conventional myosinII serving as motors. However, recent studies have demonstrated that actin can serve as tracts for long-range transport of vesicles. The current studies uncover a novel acto-Nesprin filamentous pathway, dNesp1 railroad tracks, which serve as a long-range pathway for mRNA localization and synapse maturation during development and plasticity (Packard, 2015).

Characterization of myosin-IA and myosin-IB, two unconventional myosins associated with the Drosophila brush border cytoskeleton

The expression patterns of myosin-IA (MIA) and myosin-IB (MIB), two novel unconventional myosins from Drosophila,have been characterized through immunoblot analysis and immunocytochemistry of embryos, larvae, and adults. The appearance and distribution of both proteins during embryogenesis is correlated with the formation of a brush border within the alimentary canal as documented at the ultrastructural level. MIA and MIB, both found predominantly at the basolateral domain of immature enterocytes, exhibit increased expression at the apical domain of differentiated enterocytes co-incident with microvillus assembly. Colocalization of MIA and MIB to larval and adult gut by confocal microscopy demonstrates distinct but overlapping subcellular distributions of these two proteins. In the larval brush border, MIA is enriched in the subapical terminal web domain whereas MIB is found predominantly in the apical microvillar domain. In the adult gut, MIA and MIB both exhibit a microvillar component as MIA attains a more apical position in addition to its previous terminal web locale. MIB is also found in egg chambers at both the basolateral and apical surfaces of the somatic follicle cells during oogenesis. MIA and MIB both demonstrate ATP-dependent extraction from the larval brush border cytoskeleton and exogenous F-actin, biochemical properties characteristic of functional myosins-I (Morgan, 1995).

Search PubMed for articles about Drosophila Myo61F

Gillespie, P. G., et al. (2001). Myosin-I nomenclature. J. Cell Biol. 155(5): 703-4. PubMed ID: 11724811

Hegan, P. S., Mermall, V., Tilney, L. G. and Mooseker, M. S. (2007). Roles for Drosophila melanogaster Myosin IB in maintenance of enterocyte brush-border structure and resistance to the bacterial pathogen Pseudomonas entomophila. Mol. Biol. Cell 18(11): 4625-36. PubMed ID: 17855510

Hozumi, S., et al. (2006). An unconventional myosin in Drosophila reverses the default handedness in visceral organs. Nature 440(7085): 798-802. PubMed ID: 16598258

Liehl, P., Blight, M., Vodovar, N., Boccard, F., and Lemaitre, B. (2006). Prevalence of local immune response against oral infection in a Drosophila/Pseudomonas infection model. PLoS Pathol. 2: e56. PubMed ID: 16789834

McConnell, R. E. and Tyska, M. J. (2007). Myosin-1a powers the sliding of apical membrane along microvillar actin bundles. J. Cell Biol. 177(4): 671-81. PubMed ID: 17502425

Morgan, N. S., Heintzelman, M. B. and Mooseker, M. S. (1995). Characterization of myosin-IA and myosin-IB, two unconventional myosins associated with the Drosophila brush border cytoskeleton. Dev. Biol. 172(1): 51-71. PubMed ID: 7589814

Packard, M., Jokhi, V., Ding, B., Ruiz-Canada, C., Ashley, J. and Budnik, V. (2015). Nucleus to synapse Nesprin1 railroad tracks direct synapse maturation through RNA localization. Neuron 86(4): 1015-28. PubMed ID: 25959729

Spéder, P., Adám, G. and Noselli, S. (2006). Type ID unconventional myosin controls left-right asymmetry in Drosophila. Nature 440(7085): 803-7. PubMed ID: 16598259

Tyska, M. J. and Mooseker, M. S. (2004). A role for myosin-1A in the localization of a brush border disaccharidase. J. Cell Biol. 165: 395-405. PubMed ID: 15138292

Tyska, M. J., Mackey, A. T., Huang, J. D., Copeland, N. G., Jenkins, N. A. and Mooseker, M. S. (2005). Myosin-1a is critical for normal brush border structure and composition. Mol. Biol. Cell 16: 2443-2457. PubMed ID: 15758024

Vodovar, N., Vinals, M., Liehl, P., Basset, A., Degrouard, J., Spellman, P., Boccard, F. and Lemaitre, B. (2005). Drosophila host defense after oral infection by an entomopathogenic Pseudomonas species. Proc. Natl. Acad. Sci. 102: 11414-11419. PubMed ID: 16061818

date revised: 20 June 2015

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