In fish photoreceptor cells, Myo3A concentrates at the distal ends of long, ellipsoidal actin bundles that terminate in the calycal processes (Dosé et al., 2003
). Calycal processes are actin-filled, cellular protrusions of unknown function that extend from the inner segment of both rod and cone photoreceptors to form a cup around the base of the outer segment (Nagle et al., 1986
; Arikawa and Williams, 1991
). NINAC p174, the long-form of the Drosophila
class III myosin, concentrates in rhabdomeres, microvillus-like structures that are the site of action of many of the steps in phototransduction in fly photoreceptor cells (Porter et al., 1992
). In this report, we have shown that a significant portion of GFP-Myo3A localizes to the distal ends of actin bundles within filopodia in transfected HeLa cells (). Filopodia are thin, cellular protrusions involved in cell motility and are thought to function as sensors of the local environment and as sites for adhesion and signaling (Lewis and Bridgman, 1992
; Davenport et al., 1993
). Thus, Myo3A localizes to actin filaments present within cellular protrusions in both photoreceptors and transfected HeLa cells.
A mutation in the motor active site (Shimada et al., 1997
) of Myo3A was created to test the role of motor activity in myosin III localization. This active-site mutation is predicted to abolish ATP-cleaving activity, thus creating a motor immobilized in an ATP-bound, low actin-affinity, intermediate step of the motor cycle. The motor-altered Myo3A did not localize to filopodia tips (), which suggests Myo3A moves out toward the filopodial tip on the actin bundle under its own power. Similarly, a NINAC protein lacking the complete motor domain failed to localize to the rhabdomeres, which resulted in both ERG and retinal degeneration phenotypes (Porter and Montell, 1993
). Because myosin motor activity is unidirectional and the actin bundles in both filopodia and rhabdomeres (Arikawa et al., 1990
) are oriented with their plus-ends toward the tips of the processes, these observations suggest that class III myosins are plus-end directed motors. In agreement with this observation, Komaba et al.
) recently have reported that a truncated form of human myosin IIIA has plus-end directed, F-actin translocating activity in vitro.
The presence of a kinase domain in class III myosins implies a role in cellular signaling or regulation of motor activity. A mutation in the NINAC kinase domain resulted in normal localization but an altered electroretinogram (ERG) phenotype (Porter and Montell, 1993
). In our studies, a truncated Myo3A lacking the kinase domain did not disrupt filopodia tip localization (). These observations suggest that kinase activity is not required for either motor activity or localization. Indeed, the kinase deleted-Myo3A localized more effectively to filopodia tips than the full-length construct, as seen by the elongated fluorescent pattern in the tips and reduced cytoplasmic fluorescence. This suggests that the presence of the kinase domain may inhibit Myo3A filopodia localization to some degree. Filopodia are presumably a major site of action for signal transduction cascades, because filopodial extension, retraction, and adhesion are likely to be highly regulated. The same may be true for the filopodia-like calycal processes, because they have been shown in fish photoreceptors to be dynamic cellular extensions that lengthen and shorten in response to changes in light condition (Pagh-Roehl et al., 1992
). Perhaps one of Myo3A's functions is to localize its kinase domain to calycal processes where it participates in some local signal-transduction cascade.
The tail domain of NINAC p174 is required for rhabdomeres localization and for binding to INAD, a PDZ-domain protein that functions as a scaffold for the “signalplex” in Drosophila
photoreceptors (Wes et al., 1999
). NINAC and myosin IIIA tail domains share no detectable sequence similarity, and the Myo3A tail does not contain a PDZ-binding consensus sequence (Harris and Lim, 2001
). We have shown here that the 3THDII, which is highly conserved in vertebrate myosin IIIA tails, contains an actin-binding motif, DFRXXL. The DFRXXL motif was first identified as a novel actin-binding motif present in the N-terminal domain of smooth muscle MLCK (Smith et al., 1999
). The role of this motif has not been characterized in any other protein so far. However, Smith et al.
) have noted that putative DFRXXL motifs are present in chicken smooth muscle isoform of α-actinin, in Ca2+
/calmodulin-dependent protein kinase I, and in titin. The actual role of actin binding by the myosin IIIA tail domain is unclear. An intact DFRXXL sequence is required for Myo3A tail:actin interactions both in HeLa cells () and in vitro () and for localization of full-length Myo3A to filopodia tips (). This latter observation could be explained by a loss of Myo3A motility or an inability to target to the filopodial tip. The loss of filopodial localization by GPF:M3-inactive, which has an intact tail but altered motor, suggests the change in localization is due to a lack of motility.
The tail domain of class I myosins associates with actin filaments (Lynch et al., 1986
). The three class I myosins from Acanthamoeba
and two of the five class I myosins from Dictyostelium
contain a Gly/Pro/Ala-rich domain, termed Tail Homology 2 (TH2), that binds actin filaments in vitro (Brzeska et al., 1988
; Doberstein and Pollard, 1992
; Jung and Hammer, 1994
). It is thought that tail:actin interactions could serve to recruit type I myosins onto actin filaments to aid processivity. Alternatively, a second actin-binding site could allow the type I myosins to cross-link and contract actin filaments.
It is curious that full-length myosin IIIA does not appear to interact with actin filaments other than those found bundled in cellular protrusions, despite having two actin binding sites, one in the motor and one in the tail. It is known that tropomyosin restricts the interaction of most myosins with stress fibers. However, the Myo3A tail actin-binding site is not blocked from binding stress fibers when expressed as GFP:3THDI or GFP:3THDI,3THDII, (), suggesting that the tail binds to a different site on the actin filament than do myosin motor domains. In fact, Stull and coworkers presented NMR-derived structural evidence that the DFRXXL motifs in smooth muscle MLCK bind to an actin subunit at a unique site, opposite the side where tropomyosin and myosin II bind (Hatch et al., 2001
). Perhaps in the context of the full-length Myo3A protein, actin binding by the tail domain is regulated. Indeed, a constitutively active actin-binding motif in the tail might be expected to be detrimental to translocation of a myosin. On the other hand, perhaps weak actin filament interactions mediated by the tail may support translocation by keeping myosin IIIA in close proximity to actin filaments, while the motor domain cycles on and off the actin filament during its power strokes. Such a mechanism may be advantageous for single-headed motors, which is the predicted structure of class III myosins, as they cannot move in a hand-overhand mechanism hypothesized for two-headed motors. Alternatively, the tail actin-binding site may function to limit myosin IIIA motor activity to actin bundles. The tail may interact with adjoining filaments present in actin bundles to promote efficient motor activity. Therefore, without the tail:actin interactions available in bundles, Myo3A would lack sufficient actin interactions for motility. This model would explain the apparent absence of Myo3A on the nonbundled F-actin mesh present at the cell cortex in HeLa cells and nonbundled F-actin in photoreceptors (Dosé and Burnside, 2002
Three different human MYO3A mutations have been identified that lead to nonsyndromic hearing loss (Walsh et al., 2002
). Two of the mutations truncate the MYO3A protein before the tail domain, and a third alters a splice acceptor that leads to an unstable message. Given that NINAC is required in the fly eye and that our results indicate an important role for the tail domain in Myo3A localization, it is surprising that these mutations in human MYO3A do not result in vision problems as well. This suggests Myo3A function is critical in the ear but not in the eye, that Myo3B can compensate for Myo3A function in the retina, or that different splice variants of Myo3A may be critical in the eye and the ear.
Recently, myosin X has been shown to localize to filopodial tips in HeLa cells, where time-lapse imaging revealed it undergoes both forward and rearward movements (Berg and Cheney, 2002
). Unlike Myo3A, the filopodial tip localization of myosin X did not require its tail domain. Overexpression of full-length myosin X (but not truncated forms of myosin X) in COS7 cells caused an increase in the number and length of filopodia, indicating that myosin X or its cargo may function in filopodial dynamics. We have not observed any similar changes in filopodia of either HeLa or Cos7 cells transfected with Myo3A constructs.
Given that both myosin X and myosin IIIA localize to the tips of cell protrusions, it is intriguing to speculate that myosin IIIA may function to translocate cargo out to the distal end of the calycal processes. The Myo3A cargo is unknown, but the presence of IQ motifs beyond the neck domain in myosin IIIA proteins suggests calmodulin may be one important cargo. We have demonstrated here that myosin IIIA can concentrate calmodulin in the filopodial tips in HeLa cells (). Calmodulin is a highly conserved regulatory protein that mediates a variety of calcium iondependent signaling pathways. In Drosophila
, calmodulin localization in the rhabdomeres is dependent on NINAC myosins, because mutant flies lacking the NINAC p174 did not concentrate calmodulin in the rhabdomere, and this correlated with a defect in vision (Porter et al., 1993
). Further work toward identifying and characterizing myosin IIIA cargo is imperative in order to understand the role of Myo3A in vertebrate sensory organs.