Molecular Genetic Dissection of Mouse Unconventional Myosin-VA: Tail Region Mutations

Corresponding author: Nancy A. Jenkins, ABL-Basic Research Program, P.O. Box B, Bldg. 539, Frederick, MD 21702-1201, jenkins{at}ncifcrf.gov (E-mail).

Communicating Editor: C. KOZAK

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

We used an RT-PCR-based sequencing approach to identify the mutations responsible for 17 viable dilute alleles, a mouse-coat-color locus encoding unconventional myosin-VA. Ten of the mutations mapped to the MyoVA tail and are reported here. These mutations represent the first extensive collection of tail mutations reported for any unconventional mammalian myosin. They identify sequences important for tail function and identify domains potentially involved in cargo binding and/or proper folding of the MyoVA tail. Our results also provide support for the notion that different myosin tail isoforms produced by alternative splicing encode important cell-type-specific functions.

ONE of the best-studied classes of unconventional myosins is myosin-V (MyoV). MyoV is evolutionarily well conserved and Myo5-related genes have been identified in multiple different organisms (MOOSEKER and CHENEY 1995 ; HASSON and MOOSEKER 1996 ). Class V myosins include mouse and human MyoVA (MERCER et al. 1991 ; ENGLE and KENNETT 1994 ; MOORE et al. 1995 ; ZHAO et al. 1996 ) and mouse and rat MyoVB (HASSON et al. 1996 ; ZHAO et al. 1996 ), chicken brain MyoVA (p190) (ESPREAFICO et al. 1992 ; SANDERS et al. 1992 ), and yeast MYO2 ( JOHNSTON et al. 1991 ) and MYO4 (HAARER et al. 1994 ). Class V myosins have been shown to function as actin-based molecular motors in several different motility assays and move toward the barbed ends of actin filaments (CHENEY et al. 1993 ). Purification of MyoVA from chicken brain has allowed the visualization of individual molecules by electron microscopy. As predicted from sequence data, MyoVA forms a two-headed molecule attached to a stalk that ends in a globular structure believed to represent the terminal ~400 amino acid domain (CHENEY et al. 1993 ). This C-terminal domain is generally conserved among family members, suggesting that it encodes a conserved function(s).

Studies in both yeast and vertebrates have been very informative with respect to the function of MyoV family members. The yeast MYO2 gene was identified in a screen for temperature-sensitive cell division cycle mutations and is essential for cell survival ( JOHNSTON et al. 1991 ). At the restrictive temperature, the myo2-66 mutation produces unbudded, enlarged cells with accumulated secretory vesicles. The MYO2 protein concentrates in regions of active growth, as caps at incipient bud sites and on small buds, at the mother-bud neck just before cell separation, and in mating cells as caps on shmoo tips and at the fusion bridge of zygotes (LILLIE and BROWN 1994 ). This phenotype has led to the suggestion that MYO2 is involved in organelle trafficking or membrane targeting. HIU et al. (1996) demonstrated a requirement for MYO2 in vacuolar inheritance in yeast.

MYO4 is important for mating-type switching in yeast ( JANSEN et al. 1996 ). Cells of budding yeast give birth to mother and daughter cells that differ in that only mother cells express the HO endonuclease gene and thereby switch mating types. MYO4 is one of five cytoplasmic genes identified in a genetic screen that are required for mother-specific HO expression ( JANSEN et al. 1996 ). MYO4 is not, however, a mother-specific protein. On the contrary, MYO4 accumulates in growing buds. MYO4 apparently functions by transporting factors into the bud that repress HO expression such as the recently identified zinc finger protein ASH1 ( JANSEN et al. 1996 ).

In the mouse, MyoVA is encoded by the dilute locus (MERCER et al. 1991 ). Studies of dilute mice have suggested that mouse MyoVA is important for melanosome transport in melanocytes and transport of smooth endoplasmic reticulum in cerebellar Purkinje cell dendritic spines (DEKKER-OHNO et al. 1996 ; PROVANCE et al. 1996 ; TAKAGISHI et al. 1996 ; WU et al. 1997 ).

In an accompanying manuscript (HUANG et al. 1998 ) we used an RT-PCR-based sequence approach to identify the mutations responsible for 17 viable, presumably hypomorphic, dilute alleles. Ten of the mutations mapped to the MyoVA tail and are reported here. These mutations represent the first extensive collection of tail region mutations described for any unconventional mammalian myosin and provide interesting insights into tail region function.

	MATERIALS AND METHODS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Mice:
Seven of the alleles reported in this study were generated by mutagenesis of (101/Rl x C3H/Rl) F₁ hybrid mice, six at the Oak Ridge National Laboratory, Oak Ridge, TN (Myo5a^34DENUg, Myo5a^2ENUR, Myo5a^5ENURg, Myo5a^6PXENURg, Myo5a^34ENURg, and Myo5a^33ENURf ) and one at the Medical Research Council Radiobiology Unit, Harwell, UK (Myo5a^d-105H). The remaining mutations arose spontaneously at The Jackson Laboratory, Bar Harbor, ME. The chromosomes of origin for these spontaneous alleles are as follows: C57BL/6J, Myo5a^d-J; C57BL/10SnJ, Myo5a^d-n; and C57BL/6J, Myo5a^d-n2J. Two mutations, Myo5a^2ENUR and Myo5a^33ENURf, are extinct and all analyses were performed on frozen tissues. The remaining eight mutations are maintained at the National Cancer Institute-Frederick Cancer Research and Development Center, Frederick, MD. With the exception of Myo5a^d-n2J, all mutations are propagated in the homozygous condition. Myo5a^d-n2J mice are maintained by crossing carriers to C57BL/6J-d ^v se/d ^v se mice.

Northern analysis, protein analysis, and RT-PCR sequencing were done as described in HUANG et al. 1998 .

	RESULTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

We have used an RT-PCR-based sequencing approach to identify the mutations responsible for 17 viable dilute mutations that vary in their effect on coat color and the nervous system. Seven of the mutations mapped to the MyoVA head and are reported in an accompanying manuscript (HUANG et al. 1998 ). Ten of the mutations mapped to the MyoVA tail and are described here (Table 1; Figure 1). None of the mutations mapped to the MyoVA neck. This may reflect the small target size of the neck. Alternatively, only viable mutations were sequenced in these studies, and it is possible that neck mutations are primarily homozygous lethal because of the importance of this region in light-chain binding and subsequent regulation of MyoVA activity.

View larger version (11K): In this window In a new window Download PPT slide   Figure 1. —Schematic representation of MyoVA showing the approximate location of the MyoVA tail-region mutations. The different functional domains of MyoVA, including the head, neck and tail, are indicated. The ATP-binding site and PEST regions are indicated by black rectangles, the IQ motifs by light gray rectangles, and coiled-coil domains by dark gray rectangles. Mutations below the schematic affect only coat color whereas mutations above the schematic affect both coat color and the nervous system. Splicing mutations are indicated by arrows. Exons encoding the different alternative MyoVA tail isoforms are indicated at the bottom of the figure.

Among the 10 tail mutations, seven affect only coat color: one is a d mutation, which when homozygous results in a lightened coat, and six are d^x mutations, which when homozygous cause a coat color intermediate in phenotype between wild-type and d mice. The remaining three mutations, which are designated dⁿ, cause a lightened coat (similar to d) and have a neurological disorder of varying severity (Table 1). No d^xn mutations, that is, d^x in color in combination with a neurological disorder, were identified in the tail. This was surprising given that three of seven head region mutations were from the d^xn class (HUANG et al. 1998 ). This paucity of d^xn mutations may reflect an inherent difficulty in generating this class of alleles via mutations in the tail. Alternatively, it may simply be due to random chance.

Five of the tail mutations were induced by ethylnitrosourea (ENU) (Myo5a^5ENURg, Myo5a^34DENUg, Myo5a^2ENUR, Myo5a^34ENURg, and Myo5a^33ENURf ) (Table 1). The vast majority of germline ENU-induced mutations characterized to date are caused by A:T to G:C transitions (50%) or A:T to T:A transversions (36%) (reviewed in MARKER et al. 1997 ). The same is true for the ENU-induced tail mutations where 40% (two of five) were caused by A:T to G:C transitions and 60% (three of five) by A:T to T:A transversions (Table 1).

The five other mutations mapping to the tail are either spontaneous (three), X-ray-induced (one), or induced by a combination of X-irradiation and ENU mutagenesis (one) (Table 1). The three spontaneous mutations, Myo5a^d-J, Myo5a^d-n, Myo5a^d-n2J, were caused by a genomic deletion, a missense mutation, or a splicing mutation, respectively (Table 1). All three types of lesions have been commonly associated with spontaneous mouse germline mutations (summarized in BEDELL et al. 1997 ). The X-ray-induced mutation, Myo5a^d-105H, was caused by a splicing mutation resulting from a G to T transversion (Table 1). Although X-ray-induced mutations are usually associated with large genomic deletions, inversions, or other complex rearrangements, a few X-ray-induced mutations have been associated with point mutations (MARKER et al. 1997 ). Myo5a^6PXENUe was found in the offspring of an (101/Rl x C3H/Rl) F₁ male that had received 300r X-rays five days prior to being injected with 100 mg/kg ENU. The Myo5a^6PXENUe mutation was caused by a missense mutation resulting from an A4596G transition. As ~50% of all ENU-induced mutations are caused by A:T to G:C transitions, it seems most likely that the Myo5a^6PXENUe mutation was induced by ENU. In support of this notion is the observation that Myo5a^6PXENUe was induced in spermatogonial stem cells in which 100 mg ENU/kg are more mutagenic than are 300r X rays (RUSSELL 1965 ; RUSSELL et al. 1982 ). In light of this conclusion, we have recalculated the number of transitions and transversions observed in the ENU-induced alleles and find that it is 50% for each class.

MyoVA mRNA and protein expression levels:
To determine if any of the tail mutations affected Myo5a mRNA expression levels, brain RNA from each of the 10 mutations was characterized by Northern analysis (Figure 2) and Myo5a mRNA levels were quantitated relative to a wild-type control (Table 1). Myo5a mRNA levels were as follows: not significantly altered by five mutations, Myo5a^d-105H, Myo5a^2ENUR, Myo5a^6PXENUe, Myo5a^33ENURf and Myo5a^d-n; moderately reduced by three mutations, Myo5a^5ENURg, Myo5a^34DENUg, and Myo5a^34ENURg; and severely reduced by two mutations, Myo5a^d-J and Myo5a^d-n2J.

View larger version (49K): In this window In a new window Download PPT slide   Figure 2. —Northern blot analysis of Myo5a mRNA levels from brain of homozygous viable dilute mutant mice. The three principal 7-, 8-, and 12-kb Myo5a transcripts are shown. RNA from C57BL/6J wild-type brain was used as control. Gapd (1.35 kb) was used as a loading control.

To determine if any of the mutations affected MyoVA protein levels, extracts from mutant brain were characterized by Western analysis using antibodies directed against the MyoVA head (Figure 3) and the levels quantitated relative to an internal MyoVI control (Table 1). Protein levels were severely reduced by seven mutations, Myo5a^5ENURg, Myo5a^34DENUg, Myo5a^6PXENUe, Myo5a^d-J, Myo5a^33ENURf, Myo5a^d-n, and Myo5a^d-n2J, and moderately reduced by two others, Myo5a^d-105H and Myo5a^34ENURg. Similar to what was seen for the head region mutations (HUANG et al. 1998 ), the reduction in protein levels was proportional to the severity of the mutant phenotype in only a few cases, such as Myo5a^d-n and Myo5a^d-n2J (Table 1). In contrast, the mildest allele in all of these studies, Myo5a^5ENURg, produces only 2% wild-type protein, and yet there is barely an effect on pigmentation (Table 1).

View larger version (29K): In this window In a new window Download PPT slide   Figure 3. —Western blot analysis of MyoVA protein levels from brain of homozygous viable dilute mutant mice. Extracts from mutant brain as well as wild-type brain and spleen were probed with a MyoVA head region antibody. MyoVI was used as a loading control.

Mutations in the coiled-coil domain of the tail:
Two of the Myo5a mutations, Myo5a^d-J and Myo5a^34DENUg, mapped to the coiled-coil domain of the tail that is involved in protein dimerization (Figure 1). The Myo5a^d-J mutation is unique in these studies in that Myo5a^d-J animals display the full mutant phenotype in the coat but are neurologically normal (Table 1). A combination of RNA and genomic sequence analysis indicated that the Myo5a^d-J mutation results from an ~1-kb genomic deletion that removes 8 bp of coding sequence from the first coiled-coil domain (Figure 4A). Because a splice acceptor site is removed by the deletion, no wild-type Myo5a transcripts are expressed in Myo5a^d-J mice. Three mutant splicing products, however, were detected following RT-PCR amplification and cloning of Myo5a^d-J brain mRNA. These transcripts were generated using cryptic splice acceptor sites located in both intron as well as exon sequence (Figure 4A). Two of these mutant transcripts, the 12-bp deletion and the 114-bp deletion, are in frame. Because Myo5a^d-J mice are neurologically normal it would appear that at least one of these mutant proteins is functional in brain.

View larger version (55K): In this window In a new window Download PPT slide   Figure 4. —Myo5a splicing mutations: genomic alterations and alternative splice products. Wild-type Myo5a genomic sequences are shown at the top of each panel followed by the corresponding mutant Myo5a genomic sequence. The different mutant splice products were determined by sequencing of agarose gel separated and/or cloned PCR amplification products. The relative abundance of the different splices, indicated in parentheses, was estimated by the number of clones obtained. At least 20 clones were analyzed for each mutant mouse. A deletion or insertion is indicated by letter D or I after the base pair change. Capital letters indicate exon sequence; lowercase letters indicate intron sequence. Dashes indicate noncontiguous sequence. Boxes represent cDNA sequences detected in these studies. (A) Myo5ad-J mutation. The first nucleotide shown at the far left is at position 3345 in the wild-type Myo5a cDNA; (B) Myo5ad-105H, nucleotide 4195; (C) Myo5a2ENUR, nucleotide 4195; (D) Myo5a34ENURg, nucleotide 4756; (E) Myo5ad-n2J, nucleotide 4756. The black rectangles shown in B–E represent the position and nature of the mutation.

The Myo5a^34DENUg mutation results from a T3672A transversion that introduces an L1211Q substitution into the second coiled-coil domain (Table 1, Figure 1). Computer analysis using MacVector 6.0.1, Protein Analysis Toolbox, indicates that the coiled-coil structure is not disrupted by this substitution, which is consistent with the mild d^x phenotype caused by this allele. However, this mutation does appear to destabilize the protein and possibly the mRNA as well (Table 1).

Mutations in other regions of the tail:
Tissue-specific splicing mutations: Three alternative splice forms of the MyoVA tail can be identified by PCR amplification and agarose gel electrophoresis (Figure 5). Splice form #3 is unique to brain. Splice forms #1 and #2 are seen in most other tissues, although their relative expression levels vary from tissue to tissue. Two mutations, Myo5a^d-105H and Myo5a^2ENUR, map to this alternatively spliced region and specifically affect splice form #1. The Myo5a^d-105H mutation results from a G to T transversion in exon F (Figure 4B). As a result of this mutation exon F is skipped and splice form #1 is not expressed (Figure 5). Neither splice form #2 nor #3 is affected by the exon F mutation (Figure 5). A number of investigators have reported that exon sequence is important for splicing (MARDON et al. 1987 ; HELFMAN et al. 1988 ; COOPER and ORDAHL 1989 ; HAMPSON et al. 1989 ; KATZ and SKALKA 1990 ; FU et al. 1991 ; COOPER 1992 ; STEINGRIMSDOTTIR et al. 1992 ; WATAKABE et al. 1993 ). Exon mutations could affect splicing in at least two ways. First, they could disrupt elements recognized by the splicing machinery, such as factors regulating alternative splicing. Second, they could change the secondary structure of the pre-mRNA and either sequester or expose splice sites. The precise mechanism responsible for exon F skipping in this mutant remains to be determined.

View larger version (33K): In this window In a new window Download PPT slide   Figure 5. —Tissue specific splicing mutations. The different alternative splice forms of Myo5a were amplified by PCR. The structure of the three alternative splice forms of Myo5a are shown at the bottom of the figure. The capital letters represent exons as described previously (SEPERACK et al. 1995 ). The bold arrows represent the primer set used in RT-PCR amplification. An agarose gel containing the wild-type Myo5a amplification products from brain, skin, and spleen together with products from Myo5ad-105H and Myo5a2ENUR spleen is shown at the left of the figure. Products from Myo5ad-105H and Myo5a2ENUR brain are shown at the right of the figure. The sequence of spliced forms #1 through #3 of both wild-type and mutant mice was determined by direct sequencing after gel purification of each amplified fragment.

The Myo5a^2ENUR mutation results from an A to G transition in the splice acceptor site for exon F (Figure 4C). Again, exon F is skipped and neither splice form #2 nor #3 is affected (Figure 5). This A residue is 100% conserved in all reported 3' splice junctions (PENOTTI 1991 ). The effects of mutations at this residue, however, are variable. In some cases, little effect on RNA processing is observed, whereas in others no wild-type transcripts are made (KRAWCZAK et al. 1992 ).

Although all cells contain a cytoskeleton, the cytoskeleton performs different roles and contains different components in different cell types. Tissue-specific alternative splicing is one way that a single cytoskeletal gene can produce many different proteins that perform different functions in different cell types. In the case of MyoVA, we have proposed that exon F might encode sequences necessary for MyoVA function in cells such as melanocytes and that exon B encodes sequences important for MyoVA function in the nervous system (SEPERACK et al. 1995 ). Consistent with this model, Myo5a^d-105H and Myo5a^2ENUR mice are neurologically normal; only the coat color is affected by these two mild d^x mutations (Table 1).

Non-tissue-specific splicing mutations: Two additional splicing mutations, Myo5a^34ENURg and Myo5a^d-n2J, were localized to the MyoVA tail (Figure 1). Neither mutation is tissue-specific: all forms of the protein are equally affected by these two mutations. Two classes of Myo5a transcripts are expressed in Myo5a^34ENURg mice. One class is wild type and the other class contains a 151-bp out-of-frame deletion (Figure 4D). Genomic sequence analysis showed that the 151-bp deletion results from exon skipping because of a T to C transition in the polyprimidine tract located upstream of the skipped exon (Figure 4D). As expected from the subtle nature of this mutation, as much as 80% of the Myo5a mRNA expressed in Myo5a^34ENURg mice is normally spliced. No truncated protein was detected by Western analysis (Figure 3) and wild-type protein was made at ~25% normal levels (Table 1). These results are consistent with the subtle d^x phenotype of Myo5a^34ENURg mice.

Myo5a^d-n2J is the most severe neurological mutation localized to the tail region. Like d^l mice, these mice are neurologically impaired by 14–21 days after birth. However, instead of dying from these neurological defects as mice homozygous for the d^l alleles do, mice homozygous for Myo5a^d-n2J show some improvement as they age and can be kept alive for extended periods of time. Northern and Western blot analysis showed only 12% of the normal Myo5a RNA level and <1% of the normal MyoVA protein level in Myo5a^d-n2J brain (Table 1, Figure 2 and Figure 3). Four abnormal classes of Myo5a transcripts were detected in Myo5a^d-n2J brain (Figure 4E). Two classes carried deletions and two carried insertions relative to wild-type transcripts. Genomic sequence analysis indicated that these abnormal transcripts resulted from a G to T transversion in a conserved GT splice donor consensus sequence (Figure 4E). Because the G is absolutely conserved in all GT splice donor consensus sequences, it is not surprising that wild-type Myo5a transcripts are not detected in these mice. Only two of the abnormal splices are in-frame, the 54-bp insertion and the 276-bp deletion. Although both transcripts could theoretically encode functional protein, it seems unlikely that a protein with a 92 amino acid deletion in the tail region would be functional. Presumably, however, at least one of the proteins must retain some function as homozygous Myo5a^d-n2J mice are viable.

Clustered missense mutations: Three of the globular tail region mutations were missense mutations (Table 1). Interestingly, all three mutations are located within 10 amino acids of each other (Figure 1, Table 1). Myo5a^33ENURf results from an I1510N substitution; Myo5a^5ENURg, a M1513K substitution; and Myo5a^6PXENUe, a D1519G substitution. None of the mutations significantly affect Myo5a mRNA levels, but all produce fairly dramatic reductions in MyoVA protein levels (2–4% wild-type levels) (Table 1). However, these reduced protein levels do not correlate well with the severity of the phenotypes. As mentioned previously, the Myo5a^5ENURg mutation produces a very subtle change in color yet makes only 2% wild-type protein. Both Myo5a^6PXENURg and Myo5a^33ENURf produce 4% wild-type protein but Myo5a^33ENURf has a much more severe effect on pigmentation than the other two alleles, and it also affects the nervous system (Table 1).

A protein truncation mutation:
The Myo5a^d-n mutation results from a C5558T transition that introduces a stop codon at residue 1840 of the protein (Figure 1, Table 1). Fourteen amino acid residues are deleted from the end of the mature MyoVA protein by this mutation. Myo5a mRNA levels are not significantly affected in Myo5a^d-n mice (91%); however, MyoVA protein levels are dramatically reduced (<1%) (Table 1). Myo5a^d-n is similar to the Myo5a^33ENURf mutation with respect to both color and neurological abnormalities. At 14–21 days after birth the mutant animals are as neurologically impaired as d^l mice. However, as they age they recover completely and as adults are neurologically normal. A number of explanations could account for this phenotype in Myo5a^d-n mice. For example, the 14 amino acids deleted by this mutation could encode residues important for MyoVA function in young animals but not in the adult, at least with respect to the nervous system. Alternatively, the phenotype of Myo5a^d-n mice may be explained by the very low MyoVA protein levels (<1%) seen in Myo5a^d-n mice. Perhaps higher MyoVA protein levels are required within the first few weeks after birth when the nervous system is still developing as compared to older animals, in which the nervous system is fully developed.

	DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

In studies reported here we describe 10 mutations that map to the coiled-coil or globular domains of the tail region of unconventional mouse MyoVA. These mutations represent the first extensive collection of tail region mutations described for any unconventional myosin. Although a large number of mutations have been reported for another unconventional myosin, Myo7a/MYO7A (GIBSON et al. 1995 ; WEIL et al. 1995 ; WESTON et al. 1996 ; LEVY et al. 1997 ; LIU et al. 1997 ), only two missense mutations have been mapped to the MYO7A tail (LEVY et al. 1997 ). One mutation is located in the C-terminal Ezrin-Radixin-Moesin (ERM) domain that is conserved in band 4.1 superfamily members. The presence of the mutation may impair binding of the MYOVIIA ERM domain to its putative ligand. The other mutation lies 20 amino acids downstream of the -helical domain, which is predicted to be involved in MYOVIIA dimerization.

Four of the 10 tail mutations were splicing mutations, five were missense mutations, and one resulted from an ~1-kb intragenic deletion. These results contrast with those for the head region mutations where all seven were missense mutations (HUANG et al. 1998 ). The reason for this apparent difference is not clear. It does not appear to be because of the nature of the mutagen responsible for these mutations because all seven head region mutations were chemically induced as were two of four splicing mutations identified in the tail.

The Myo5a head mutations were roughly equally distributed along the length of the MyoVA head, and there was not obvious clustering of mutations with respect to phenotypic class, suggesting a simple model where the least severe amino acid changes correlate with the least severe dilute alleles (d^x), and the most severe are d^xn or dⁿ (HUANG et al. 1998 ). With the exception of the two tissue specific splicing mutations, Myo5a^d-105H and Myo5a^2ENUR, tail region results were consistent with this simple model.

The results for several tail region mutations suggest that the nervous system is relatively insensitive to the level of MyoVA protein. For example, mice homozygous for the Myo5a^d-J mutation show a fully diluted coat color but are neurologically normal at all ages. The mutation is caused by a ~1-kb genomic deletion in the coiled-coil region, which decreases the level of protein in brain to <1% of that found in normal mice. Furthermore, none of the protein is wild type. Assuming that the region encompassed by the deletion is necessary for both melanocyte and neuron function, then a remarkably small amount of MyoVA is sufficient to rescue neurological phenotype. Furthermore, a nonsense mutation in Myo5a^d-n mice results in the production of a 14 amino acid C-terminal truncated protein that is expressed at <1% of the level of that found in wild-type brain. Although Myo5a^d-n mice are neurologically impaired when young, as adults they are normal and even breed in the homozygous condition.

Studies in both the dilute rat and the dilute mouse suggest that MyoVA is necessary for the transport of smooth endoplasmic reticulum (SER) to the dendritic spines of cerebellar Purkinje cells (DEKKER-OHNO et al. 1996 ; TAKAGISHI et al. 1996 ). Purkinje cell dendritic outgrowth begins shortly after birth, at a time when the neurological phenotype of dilute mice is first observed and is completed by 30 days of postnatal development (ITO 1984). The cerebellum is the major organ within the central nervous system involved in motor control, and the neurological defects observed in dilute mice are consistent with this SER defect. The phenotype of Myo5a^d-n (as well as some of the other alleles that show improvement in neurological behavior with age) mice could reflect an increased need for MyoVA during postnatal development when Purkinje cell dendritic spines are being elaborated. Perhaps the low level of truncated MyoVA protein expressed in Myo5a^d-n mice is sufficient for some SER transport, which rescues the lethality associated with dilute null alleles. Once dendritic outgrowth ceases in Myo5a^d-n mice, SER transport could conceivably continue and eventually fill the empty dendritic spines that may have been produced during the neonatal growth of Myo5a^d-n mice.

Two of the most informative tail region mutations were Myo5a^d-105H and Myo5a^2ENUR. Both are tissue-specific splicing mutations that delete one of the three MyoVA splice forms. The splice form that is missing contains exon F, which is expressed in tissues such as spleen and skin but not in brain. This pattern of expression suggests that exon F might encode sequences that allow MyoVA to carry different cargoes in tissues such as skin (melanocytes) and spleen versus brain (Purkinje cells). Consistent with this model, Myo5a^d-105H and Myo5a^2ENUR mice are neurologically normal: only the coat color is mildly affected by these two mutations. In the future it will be interesting to perform yeast two-hybrid screens using tails as baits that either contain or lack exon F. Perhaps it will be possible to identify proteins expressed in tissues such as melanocytes that only bind baits carrying exon F.

Another region that may function in cargo binding is suggested by three mutations that cluster within 10 amino acids in the MyoVA tail, Myo5a^33ENURf, Myo5a^5ENURg, and Myo5a^6PXENUe. This clustering is not expected by random chance (P < 1.6 x 10^-7), suggesting that an important functional domain of Myo5a may be located in this region of the tail. Interestingly, this 10 amino acid region is located 8 amino acids upstream from an ~280 amino acid C-terminal region that shares extensive sequence homology with two genes, AF-6 and cno (PONTING 1995 ). AF-6 was originally identified as the fusion partner for ALL-1 in acute myeloid leukemias carrying the t(6;11) chromosome translocation (PRASAD et al. 1993 ). cno is a Drosophila melanogaster gene thought to mediate interactions between the Notch signaling cascade and other signaling pathways (MIYAMOTO et al. 1995 ). What makes this homology interesting is that, in addition to the myosin homology, both AF-6 and cno also share a region of homology with the kinesin-like molecule unc-104 (PONTING 1995 ). Like MyoVA, unc-104 has been proposed to function in anterograde transport within neurons (HALL and HEDGECOCK 1991 ). Although the function of the domains in AF-6 and cno showing homology to kinesins and myosins is unknown, it has been proposed that when these domains are within intact motor proteins, they may mediate binding to cargo molecules and that, when within AF-6 and cno, they may possess similar binding functions (PONTING 1995 ). Perhaps the three Myo5a mutations that are located near this AF-6/cno homology domain interfere with this cargo binding in melanocytes (Myo5a^5ENURg and Myo5a^6PXENUe) and melanocytes and neurons (Myo5a^33ENURf ).

An alternative explanation for this clustering of mutations is that this region of the tail is important for protein folding. Consistent with this explanation, computer analysis (MacVector 6.0.1; Protein Analaysis Toolbox) predicts that all three mutations are not surface exposed and two of the mutations, Myo5a^33ENURf and Myo5a^5ENURg, are located in hydrophobic regions. The third mutation, Myo5a^6PXENUe, is predicted to be located at the border between a hydrophobic and hydrophilic region. In addition, all three mutations are predicted to increase the hydrophilicity of the protein. An effect on protein folding could also account for the protein instability that results from these three mutations.

Finally, recent studies have identified mutations in MYOVA in humans with Griscelli disease (PASTURAL et al. 1997 ). Griscelli disease is a rare autosomal recessive disorder characterized by pigment dilution, variable cellular immunodeficiency and onset of acute phases of uncontrolled lymphocyte and macrophage activation, leading to death in the absence of bone-marrow transplantation. The pigment defect in Griscelli patients is associated with clumping of pigment in the hair shaft and an accumulation of melanosomes within melanocytes similar to what is seen in dilute mice. However, unlike dilute mice, Griscelli disease has many features in common with the Chédiak-Higashi syndrome (CHS) including oculocutaneous albinism and immunodeficiency, as well as the occurrence of uncontrolled lymphoxyte/macrophage activation syndrome. CHS patients have defects in a gene called LYST, which is thought to play a role in vesicular transport and intracellular protein trafficking (BARBOSA et al. 1996 ; NAGLE et al. 1996 ). The phenotypic similarity between Griscelli and CHS patients suggests that LYST and MYOVA proteins may directly interact or function in the same intracellular trafficking pathway.

Two MYOVA mutations have been identified in Griscelli patients (PASTURAL et al. 1997 ). One mutation is in the coil-coil region of the tail and probably affects protein dimerization. The second mutation introduces a stop codon in front of the putative calmodulin/light chain-binding domain and likely represents a null allele. Interestingly, the patient with the stop codon has a severe neurological disorder, similar to the neuroectodermal melanolysosomal disease described by ELEJALDE et al. 1977 ; and the patient with the tail region mutation is neurologically normal. It thus appears that in humans, as in mice, only the more severe MYOVA mutations have a neurological phenotype. Human MYOVA null alleles are not always neurologically lethal, however, although some Griscelli patients have been reported to die from brain degeneration associated with cellular infiltration (HARALDSSON et al. 1991 ; HURVITZ et al. 1993 ). It is possible that some other motor protein can compensate for the absence of MYOVA in the human, but not mouse, nervous system.

	ACKNOWLEDGMENTS

We thank JO PETERS and BRUCE CATTANACH (MRC Radiobiology Unit, Harwell, UK) for providing mice carrying the Myo5a^{d -105H} allele and LINDA WASHBURN and HOPE SWEET (The Jackson Laboratory, Bar Harbor, ME) for providing mice carrying the Myo5a^{d -J}, Myo5a^{d -n}, and Myo5a^{d -n2J} alleles. We are grateful to DEBORAH A. SWING and MARILYN POWERS for their help with these studies. This research was supported by the National Cancer Institute, Department of Health and Human Services, under contract with ABL (N.G.C. and N.A.J.); a basic research grant from the Muscular Dystrophy Association and National Institutes of Health grant DK-25387 (M.S.M.); American Cancer Society postdoctoral fellowship PF-4316 to V.M.; and the Office of Health and Environmental Research, U.S. Department of Energy (under contract DE-AC05-96OR22464 with Lockheed Martin Energy Research Corp.) jointly with the National Institute of Environmental Health Sciences under Interagency Agreement No. 1-Y 01-ES-50318-00.

Manuscript received September 19, 1997; Accepted for publication December 23, 1997.

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