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).

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 Citation: 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 Citation: 17855510

Hozumi, S., et al. (2006). An unconventional myosin in Drosophila reverses the default handedness in visceral organs. Nature 440(7085): 798-802. PubMed citation: 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 Citation: 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 Citation: 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 Citation: 7589814

Spéder, P., Adám, G. and Noselli, S. (2006). Type ID unconventional myosin controls left-right asymmetry in Drosophila. Nature 440(7085): 803-7. PubMed citation: 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 Citation: 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 Citation: 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 Citation: 16061818

date revised: 20 June 2009

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