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Human Molecular Genetics Advance Access originally published online on September 26, 2007
Human Molecular Genetics 2007 16(24):3160-3173; doi:10.1093/hmg/ddm279
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© The Author 2007. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

An MYH9 human disease model in flies: site-directed mutagenesis of the Drosophila non-muscle myosin II results in hypomorphic alleles with dominant character

Josef D. Franke1, Ruth A. Montague1, Wayne L. Rickoll2 and Daniel P. Kiehart1,*

1 Department of Biology, DCMB Group, Duke University, Durham, NC 27708-0338, USA and 2 Department of Biology, University of Puget Sound, Tacoma, WA 98416, USA

* To whom correspondence should be addressed at: Department of Biology, DCMB Group, Rm. 4330 French Family Science Center, Science Drive, Duke University, PO Box 90338, Durham, NC 27708-0338, USA. Tel: +1 9196138157; Fax: +1 9196138177; Email: dkiehart{at}duke.edu

Received June 18, 2007; Accepted September 20, 2007


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 NOMENCLATURE
 FUNDING
 REFERENCES
 
We investigated whether or not human disease-causing, amino acid substitutions in MYH9 could cause dominant phenotypes when introduced into the sole non-muscle myosin II heavy chain in Drosophila melanogaster (zip/MyoII). We characterized in vivo the effects of four MYH9-like mutations in the myosin rod—R1171C, D1430N, D1847K and R1939X—which occur at highly conserved residues. These engineered mutant heavy chains resulted in D. melanogaster non-muscle myosin II with partial wild-type function. In a wild-type genetic background, mutant heavy chains were overtly recessive and hypomorphic: each was able to substitute partially for endogenous non-muscle myosin II heavy chain in animals lacking zygotically produced heavy chain (but the penetrance of rescue was below Mendelian expectation). Moreover, each of the four mutant heavy chains exhibits dominant characteristics when expressed in a sensitized genetic background (flies heterozygous for RhoA mutations). Thus, these zip/MyoIIMYH9 alleles function, like certain other hypomorphic alleles, as excellent bait in screens for genetic interactors. Our conjecture is that these mutations in D. melanogaster behave comparably to their parent mutations in humans. We further characterized these zip/MyoIIMYH9 alleles, and found that all were capable of correct spatial and temporal localization in animals lacking zygotic expression of wild-type zip/MyoII. In vitro, we demonstrate that mutant heavy chains can dimerize with endogenous, wild-type heavy chains, fold into coiled-coil structures and assemble into higher-order structures. Our work further supports D. melanogaster as a model system for investigating the basis of human disease.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 NOMENCLATURE
 FUNDING
 REFERENCES
 
MYH9 encodes one of the three non-muscle myosin II heavy chains in humans (MYH9 encodes IIA, MYH10 encodes IIB and MYH14 encodes IIC). Mutations in MYH9 cause several autosomal dominant, giant-platelet disorders that are collectively known as MYH9-related disorders (1–7). Virtually all individuals with this disorder exhibit platelet macrocytosis (enlarged platelets), thrombocytopenia (reduced platelet number) and leukocyte inclusions. Mutations in MYH9 also cause less penetrant, non-hematologic manifestations—high-tone, sensorineural deafness; cataracts; and nephritis. The severity and occurrence of these symptoms vary among individuals, can be life threatening and are likely due to the effects of variable genetic backgrounds (e.g. they have polymorphic loci that genetically interact with MYH9). Identification of such polymorphic loci is extremely difficult and time intensive to study in humans. The development of a disease model in a genetically tractable organism with fast generation time, facile genetic screening approaches and straightforward identification of interacting loci could prove extremely useful in the identification of factors that contribute to the variable clinical manifestations. Further, the ability to screen potential drug and therapeutic treatments in a model organism is very attractive.

Functional myosin II heavy chains, including those encoded by MYH9, are normally incorporated into heterohexameric motor molecule complexes, which consist of two heavy chains and two pairs of light chains (Fig. 1A). Each myosin II heavy chain can be divided into three regions or domains: a globular head or motor domain, which is responsible for actin binding and ATP hydrolysis; a neck region, which is important for regulation, light chain binding and functions as a lever arm critical for chemomechanical force production; and a rod, or tail, region, which is responsible for dimerization and filament formation. Native myosin II heterohexamers self-assemble into bipolar, or side-polar, filaments in order to generate forces for the displacement of antiparallel actin filaments. Myosin II filament assembly is specified and mediated by sequences in the rod region (8–12) and requires at least two conceptually distinct steps (that may overlap temporally). First, a heptad repeat mediates the dimerization of individual heavy chains by correctly folding the nascent polypeptide chains into the {alpha}-helical coiled-coil structure, which constitutes the extended rod-like tail that characterizes all myosin IIs. Second, charge distributions along the length of each coiled-coil dimer ensure that correct lateral associations occur such that individual myosin molecules align in proper register and orientation to form functional side polar filaments under physiological salt conditions [at high salt, filaments do not assemble (9,10)].


Figure 1
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  		Figure 1. Alignments of the D. melanogaster non-muscle myosin II and all human myosin II heavy chains. (A) Schematic of a native myosin II molecule showing both heavy chains and both pairs of light chains (essential light chain, ELC, and regulatory light chain, RLC, are indicated by arrows). The arrowhead marks the junction in the rod region separating subfragment 2 and light meromyosin. The bounded arrow indicates the tail fragment (amino acid 1109–1972) of the wild-type zip/MyoII rod used for in vitro studies. Asterisks mark the relative position of the four mutations studied. (B–E) Alignments of the sole D. melanogaster non-muscle myosin II (D.m. zip) and all human (H.s.) myosin II heavy chains (MYH9 = non-muscle myosin IIA, MYH10 = non-muscle myosin IIB, MYH14 = non-muscle myosin IIC, MYH1 = adult skeletal muscle myosin IIx/d, MYH2 = adult skeletal muscle myosin IIa, MYH3 = embryonic skeletal muscle myosin II, MYH4 = adult skeletal muscle myosin IIb, MYH6 = alpha cardiac muscle myosin II, MYH7 = beta cardiac muscle myosin II, MYH8 = perinatal skeletal muscle myosin II, MYH11 = smooth muscle myosin II and MYH13 = extraocular muscle myosin II). Alignments show the ten amino acids flanking either side of the amino acid residues of interest (B = R1171C, C = D1430N, D = D1847K and E = R1939X). In parenthesis, to the right of each alignment, is the relative position of this residue in the respective heavy chain. The amino acid of interest in the alignment is boxed in each to highlight the high degree of amino acid conservation in the different myosin II heavy chains. The R1939 residue lies beyond the highly conserved coiled-coil region of the rod and lies in the small, globular C-terminus (a region that often lacks a high degree of conservation). Nevertheless, the alignment shows that this residue is well conserved between the D. melanogaster non-muscle myosin II and all three human non-muscle myosin II heavy chain (MYH9, MYH10 and MYH14).

 
Disease-causing mutations in MYH9 (as well as mutations in other myosin II heavy chains causing disease) are present in sequences that span the entire heavy chain. About 75% of MYH9-related disorder families have mutations in the rod region (13). Despite the large number of mutations identified in the rod, 88% of all MYH9 rod mutations occur at four residues [amino acids 1165, 1424, 1841 and 1933 (1,6,13,14)]. These include identical mutations in unrelated families and different amino acid changes at the same amino acid residue (e.g. amino acids 1165 and 1424, specifically R1165 to C or L and D1424 to N, H or Y). Together, this suggests that these mutations cause highly specific disruptions of non-muscle myosin IIA structure and/or function and have arisen as the consequence of independent mutagenic events.

The specific biochemical mechanisms that account for how these mutations disrupt myosin II structure and/or function to cause disease are incompletely understood. Two mechanisms have been proposed for MYH9-related disorder mutations. The first proposes that mutant heavy chains are unstable, are degraded by the cell, and the observed hematologic manifestations are the result of haploinsufficiency (15,16). The second possibility is that mutant heavy chains have a dominant activity and inhibit the ability of wild-type heavy chain to perform cellular functions at wild-type levels. This could occur by (i) disrupting the assembly of bipolar filaments, (ii) impairing the ability of bipolar filaments to properly translocate actin filaments or (iii) by sequestering wild-type heavy chain away from sites of activity.

Using recombinant approaches, Sellers and colleagues showed that heavy meromyosin (HMM) fragments, which include the catalytic head and light chain binding domains, containing either of two MYH9 head mutations (N93K and R702C) have impaired enzymatic activity and that their ability to translocate actin filaments in vitro is significantly reduced compared with wild-type (17). Similar in vitro experiments on HMM fragments containing mutations analogous to the N93K and R702C MYH9 mutations engineered in the human MYH10 (non-muscle myosin IIB; N97K and R709C respectively) and mouse Myh14 (non-muscle myosin IIC; R722C and R730C respectively) resulted in reduced enzymatic activity and actin filament sliding (18). Precisely how reduced enzyme activity and actin filament sliding causes a dominant pathology is not known, but it is reasonable to suspect that impaired myosin head function in a heterologous filament that contains both wild-type and mutant heads might compromise myosin driven contractility in a dominant fashion.

Using recombinant approaches, we previously showed that the four most common rod mutations (R1165C, D1424N, E1841K and R1933X) were found to directly affect assembly of tail fragments into higher-ordered assemblies by one of two distinct mechanisms (19). Two mutants (R1165C and E1841K) had decreased folding into a wild-type coiled-coil structure while two (D1424N and 1933X) had intact coiled-coil structure, but native myosin II dimers were unable to laterally associate properly to form wild-type higher-order structures (19). The effect that mutations analogous to those in the MYH9 rod that cause disease have on other myosin II heavy chains remain unknown and are the subject of this investigation.

Mutations in other human conventional myosin (i.e. myosin II) heavy chains, including skeletal, cardiac and non-muscle isoforms, are responsible for a number of autosomal dominant pathologies [Table 1, see also On Mendelian Inheritance in Man database (20)]. Interestingly, only one mutation in MYH9 (amino acid 702) had an analogous mutation in a different myosin II heavy chain associated with pathology in humans (amino acid 726 in MYH14). It remains unknown if this or other mutations in myosin II that cause human pathology have conserved detrimental effects on myosin function (i.e. disrupts the function of other myosin II heavy chains).


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  		Table 1. Pathologies associated with mutations in different human myosin II heavy chains

 
In the model metazoan Drosophila melanogaster, each non-muscle myosin II polypeptide is encoded by a single gene (zipper encodes the heavy chain, zip/MyoII; spaghetti squash encodes the regulatory light chain, sqh/RLC; and mlc-c encodes the essential light chain, mlc-c/ELC) thereby simplifying the phenotypic analysis of mutations in non-muscle myosin II in D. melanogaster (21–23). As in other metazoans, functional non-muscle myosin II is necessary in D. melanogaster for cytokinesis, proper subcellular localization of cell-fate determinants during certain asymmetric cell divisions and numerous morphogenic movements during development (22,24–27). Interestingly, certain post-embryonic, lethal alleles of the zipper locus (zipEbr, zip6.1 and zip2.1) but not strong embryonic lethal alleles (i.e. molecularly null alleles, zip1 and zip2) exhibit dominant characteristics in specific genetic backgrounds [termed second-site non-complementation (28,29)]. The most extensively studied allele is zipEbr, which produces a stable heavy chain with a single amino acid substitution (R276H, 28). The zipEbr allele generally behaves as a recessive lethal (e.g. zipEbr/zipEbr animals die post-embryonically, prior to eclosion of adult flies and zipEbr/ zipwild type animals are viable with no observable phenotypes in an otherwise wild-type genetic background). However, dominant visible and semi-lethal phenotypes (progeny of the appropriate genotype are present at a frequency that is significantly less than Mendelian expectation) are observed in animals that are heterozygous for both zipEbr and mutations in certain other genes that contribute to zip/MyoII function (such as RhoA: i.e. animals whose two second chromosomes have the following genotype: RhoAwild type zipEbr/ RhoAmutant zipwild type). Thus, specific point mutations in zip/MyoII can result in an allele in which dominant phenotypes are only observed when placed in particular genetic backgrounds. These experimental findings are similar to proposed mechanisms that could explain why certain individuals with an MYH9 mutation also exhibit potentially life-threatening non-hematologic manifestations: some feature(s) of an individual's genetic background, in combination with a mutation in MYH9 results in deafness, nephritis or cataracts. The ability to identify those loci that interact with MYH9 mutations to cause these manifestations will enable a more complete understanding of the molecular basis of the phenotypes observed in MYH9-related disorders.

Here, we investigate the effects of four MYH9 disease-causing, rod mutations on the function of the sole non-muscle myosin II heavy chain in D. melanogaster. In a wild-type genetic background, these engineered mutant heavy chains result in D. melanogaster non-muscle myosin II alleles that are completely recessive, hypomorphic and encode proteins that localize correctly, both spatially and temporally, even in animals that lack zygotic expression of wild-type zip/MyoII. Nevertheless, they display dominant phenotypes in specific genetic backgrounds indicating that they function, like certain other hypomorphic alleles of zip/MyoII, as excellent bait in screens for genetic interactors (28,30). In vitro, mutant heavy chains fold into a wild-type coiled-coil structure, dimerize with endogenous, wild-type heavy chains and assemble into higher-order structures. Together, our observations suggest a mechanism by which MYH9 mutations display a diversity of distinct pleiotropic phenotypes in the human population.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 NOMENCLATURE
 FUNDING
 REFERENCES
 
MYH9-like mutant zip/MyoII heavy chain transgenes
To explore the use of D. melanogaster as a model system for MYH9-related disorders, we constructed transgenes in which highly conserved rod residues in its sole non-muscle myosin II heavy chain (zip/MyoII) were replaced with analogous mutant residues that cause MYH9-related disorders in humans. We previously generated an N-terminal GFP-tagged full-length zip/MyoII transgene (GFP-zip/MyoIIWT) whose product is functional and can substitute for endogenous zip/MyoII in animals lacking zygotic zip/MyoII (25). Site-directed mutagenesis was performed on GFP-zip/MyoIIWT to generate the following MYH9-like rod mutations: R1171C (corresponds to R1165C in MYH9, Fig. 1B), D1430N (corresponds to D1424N in MYH9, Fig. 1C), D1847K (corresponds to E1841K in MYH9, Fig. 1D) and R1939X (corresponds to R1933X in MYH9, Fig. 1E). Except for R1933X, each altered residue is included in a highly conserved region that is easily identifiable when comparing the primary sequence of MYH9 (as well as all other human myosin II heavy chains) to that of the zip/MyoII protein. In contrast, R1939X truncates the zip/MyoII heavy chain in its globular tail region, which is generally not well conserved. Nevertheless, using default conditions, alignments show that R1939 is well conserved between the D. melanogaster non-muscle myosin II and all three human non-muscle myosin II heavy chains (MYH9, MYH10 and MYH14; Fig. 1E) so that an appropriate truncation could be constructed. We refer to these mutant heavy chains as GFP-zip/MyoII R1171C, D1430N, D1847K or R1939X, the P-element mediated, white+ marked-transgenes that encode them as P[UAS-GFP-zip/MyoII R1171C, D1430N, D1847K or R1939X], or simply as GFP-zip/MyoII mutant transgenes as is appropriate. These constructs were expected to have dominant effects when expressed, so we used the bipartite Gal4-UAS transgene expression system to enable control of transgene expression levels and tissue-specific expression (31).

MYH9-like mutant zip/MyoII heavy chain transgenes do not have dominant characteristics in an otherwise wild-type genetic background
We examined flies that express a single, GFP-zip/MyoII mutant heavy chain, in otherwise wild-type animals, to see if they had observable phenotypes. No lethality was observed in animals ubiquitously expressing each mutant heavy chain and adult flies were phenotypically indistinguishable from wild-type animals (data not shown).

Dominant alleles often function in a dose-dependent manner so we also examined the biological effect of ubiquitous expression of GFP-zip/MyoII mutant heavy chains in a non-muscle myosin II heterozygous background (i.e. P[UAS-GFP-zip/MyoIIwild type OR mutant], zip1/zipwild type, see Table 2). Heterozygous animals expressing each mutant heavy chain completed development and eclosed as adult flies simultaneously with their wild-type siblings (~12 days after egg lay at 25°C; Table 2). Moreover, all such flies eclosed regardless of the GFP-zip/MyoII mutant transgene that they were expressing, and were phenotypically indistinguishable from those expressing GFP-zip/MyoIIWT. Together, these data indicate that none of the mutant heavy chains have strong or moderate dominant activity in an otherwise wild-type or heterozygous zip/MyoII genetic background. We cannot rule out that some minor, dominant effects might occur when mutant heavy chains are expressed in these animals and that the assays we used were not sufficiently sensitive to detect them. For example, our phenotypic inspection of whole flies would miss minor but real hematological defects in flies that would be comparable to the hematological defects seen in MYH9-related disorder patients.


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  		Table 2. MYH9-like zip/MyoII mutant heavy chains lack dominant negative activity and, instead, have partial wild-type function

 
MYH9-like mutant zip/MyoII heavy chain transgenes are recessive alleles of zip/MyoII
To more completely characterize these MYH9-like mutations in zip/MyoII, we examined the effect of ubiquitously expressing each GFP-zip/MyoII mutant heavy chain in homozygous zip/MyoII mutant animals. These animals, hereafter referred to as zip/MyoII-deficient animals, cannot produce zip/MyoII zygotically from their endogenous, zip/MyoII loci. In the absence of a zip/MyoII transgene, embryos homozygous for zygotic null, or near null, zip/MyoII alleles (zip1 or zip2), or that are transheterozygous for those alleles (zip1/zip2), display aberrant developmental phenotypes midway through embryogenesis (~12 h after egg lay during dorsal closure or head evolution stages). All fail to properly complete embryogenesis and do not hatch as larvae (32). The maternal load of zip/MyoII protein during oogenesis is sufficient for the successful progression of earlier embryonic development without zygotic transcription of zip/MyoII.

We found that all MYH9-like zip/MyoII mutant heavy chains had some wild-type activity—they were able to rescue the early, embryonic lethal phenotype at a frequency comparable to a wild-type, GFP-zip/MyoII transgene (GFP-zip/MyoIIR1171C = 80 ± 8%, GFP-zip/MyoIID1430N = 93 ± 4%, GFP-zip/MyoIID1847K = 88 ± 4% and GFP-zip/MyoIIR1939X = 91 ± 3%; compared to rescue with the wild-type transgene; GFP-zip/MyoIIWT = 92 ± 2%; Table 3). In addition, many rescued larvae went on to complete subsequent larval and pupal development. Based on these results, we concluded that all of the examined MYH9-like rod mutations in zip/MyoII produced stable heavy chains that lacked any direct dominant negative activity. These rescue results also demonstrate that the sqh-Gal4 driver (sqh encodes the regulatory light chain of zip/MyoII), the only non-muscle myosin II Gal4 driver available, was appropriate for ubiquitous expression. We also assayed the relative amount of each GFP-zip/MyoII heavy chain present in rescued animals compared to the amount of endogenous zip/MyoII in wild type animals (see Supplementary Material). We found that GFP-zip/MyoII heavy chain expression was similar, or greater than, endogenous zip/MyoII protein levels.


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  		Table 3. MYH9-like mutations in zip/MyoII rescue embryonic lethality

 
Interestingly, three of the GFP-zip/MyoII mutant heavy chains (D1430N, D1847K or R1939X) failed to rescue later developmental stages (adult eclosing flies) at frequencies comparable to the GFP-zip/MyoIIWT control (Table 3). To verify these findings, we performed similar rescue analysis on different P-element insertions of the GFP-zip/MyoIID1430N or D1847K transgenes and found similar results (for both D1430N and D1847K, none of the 150 selected embryos went on to eclose as adult flies).

We also found that the overall rate of development was reduced in animals rescued with any GFP-zip/MyoII mutant heavy chain compared to those animals rescued with GFP-zip/MyoIIWT. Animals rescued with mutant heavy chains generally began pupation 1 day later than GFP-zip/MyoIIWT rescued animals and when and if adult flies did eclose they did so at least 2 days later than GFP-zip/MyoIIWT rescued animals.

Thus, GFP-zip/MyoII mutant heavy chains only partially rescue zip/MyoII-deficient animals (Table 3) and behave as partial, loss of function, recessive alleles. They lack severe, and overt, dominant phenotypes when expressed in wild-type and heterozygous heavy chain backgrounds (Table 2).

MYH9-like zip/MyoII heavy chains function as specialzip/MyoII alleles that can be used to identify secondsite, non-complementing (SSNC) loci
We next examined whether or not these zip/MyoIIMYH9 alleles resulted in a sensitized genetic background that enable the in vivo identification of genetically interacting loci. We found that each MYH9-like zip/MyoII heavy chain exhibited second-site non-complementation (Table 4). Each GFP-zip/MyoII mutant heavy chain was expressed in a genetic background that was designed to mimic the genotypes we and others originally used to demonstrate second site non-complementation of both zipEbr and zip6.1 with RhoA (28,30 and references therein). Each zip/MyoIIMYH9 heavy chain allele was expressed using engrailed-Gal4 (en-Gal4) in a background that was heterozygous both for a null allele of zip/MyoII (e.g. endogenous zip/MyoII loci were zip2/zipwild type) and for a strong RhoA allele (i.e. RhoAE3.10/RhoAwild type). This Gal4 was used because the engrailed promoter drives expression at high levels throughout D. melanogaster development and is specific to particular tissues and cells (e.g. the posterior, but not the anterior, compartment of the wing) that have been well characterized. Such tissue-specific expression provides a useful internal control within each individual animal, permitting verification that phenotypes are observed only in the cells in which the transgene is expressed.


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  		Table 4. MYH9-like zip/MyoII heavy chains display dominant phenotypes in specific genetic backgrounds and function as second-site non-complementation alleles

 
Clear phenotypes were apparent when MYH9-like zip/MyoII mutant heavy chains were expressed in animals that were transheterozygous for both RhoA and zip (P[UAS-GFP-zip/MyoII mutant], RhoAwild typezip2/engrailed-Gal4, RhoAE3.10 zipwild type; see Table 4; Fig. 2). Some animals had both malformed legs and wings while others showed only malformed wing phenotypes. For several reasons, we believe that the phenotypes are the result of a specific genetic interaction with MYH9-like heavy chains. First, these phenotypes were absent when mutant heavy chains were similarly expressed in a RhoA wild-type background (Table 4), demonstrating that phenotypes were specific to the sensitized, RhoA/+ genetic background and not simply the result of expression of GFP-zip/MyoII mutant heavy chains. Second, phenotypes were only observed in those tissues or cells expressing the transgene (engrailed-expressing; below the dotted line indicated in Fig. 2A).


Figure 2
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  		Figure 2. Dominant wing and leg phenotypes occur when MYH9-like zip/MyoII heavy chain are expressed in a RhoA/+ background. (A–D) MYH9-like zip/MyoII mutant heavy chains were specifically expressed in the posterior compartment of wing imaginal discs using engrailed-Gal4. Such expression results in each wing having an experimental region, the posterior compartment (below the dotted line) and an internal control, the anterior compartment (above the dotted line). The dashed line indicates the tissue-specific boundary of the engrailed promoter in the adult wing. Importantly, all cells comprising the entire wing are RhoA/+. The severity of observed phenotypes ranged from mild to severe. (A) Image of a wild-type wing. (B) Example of a mild wing phenotype where ectopic patterning of veins and crossveins are observed. (C) Example of a moderate wing phenotype where a portion of the posterior compartment (arrowhead) of the wing has failed to form. (D) Example of a severe wing phenotype where a significant amount of the posterior wing compartment tissue has been lost. Importantly, all observed phenotypes were restricted to the compartment where each mutant heavy chain was expressed. The control, anterior compartment was indistinguishable from wild type in each example. (E–H). engrailed promoter expression of MYH9-like zip/MyoII heavy chains in the leg caused mild to severe leg malformations. (E) Image of a wild-type leg, which consists of tarsal segments, the tibia and the femur. (F) Example of a mild leg phenotype where morphological defects are observed in each of the three leg components. (G and H) Examples of severe phenotypes where dents (arrowheads) and twisting of the femur are most noticeable. The tibia and tarsal segments also display malformed phenotypes. Scale bar in A and E is 200 µm and apply to A–D and E–H, respectively.

 
Importantly, both the penetrance and expressivity (extent of malformation of the legs and wings) of phenotypes when MYH9-like zip/MyoII mutant heavy chains were expressed were in all cases greater than when GFP-zip/MyoIIWT was expressed. The incidence of malformed flies when GFP-zip/MyoIIWT was expressed in a RhoA background (4%) is comparable to the rate of malformed flies observed in heterozygous RhoAE3.10 flies with a wild-type zip background (see Table 1 in 30). In these GFP-zip/MyoIIWT expressing flies, only mild malformations of the wings and legs were observed (similar to Fig. 2B and F). In comparison, much more severe wing and leg phenotypes (similar to Fig. 2C, D, G and H) were observed when each MYH9-like mutant heavy chain was similarly expressed. Therefore, both the penetrance of malformed flies and the expressivity of the phenotypes were increased when MYH9-like zip/MyoII mutant heavy chains were expressed. The malformed wing and leg phenotypes observed are also consistent with phenotypes we have observed when zip/MyoII function was specifically perturbed using other zip/MyoII constructs (e.g. the zip/MyoII tail or the zip/MyoII neck and tail) that were designed and shown to function as dominant negative alleles (Manuscript in preparation and 25).

In sum, we conclude that these MYH9-like zip/MyoII transgenes function as special alleles of zip/MyoII that exhibit second-site non-complementation characteristics in trans to heterozygosity in certain, other loci.

MYH9-like zip/MyoII heavy chains undergo correct spatial and temporal localization
To understand how these mutations in zip/MyoII have the observed biological effects, we ascertained whether or not the mutant heavy chains localize in the correct spatial and temporal distribution. We examined the subcellular localization of each GFP-zip/MyoII mutant heavy chain compared to GFP-zip/MyoIIWT during the dorsal closure stage of embryogenesis (Fig. 3). To reduce the contribution of endogenous zip/MyoII that remains at dorsal closure stages, we examined GFP-zip/MyoII mutant heavy chains in embryos that were zygotically deficient for zip/MyoII and contain only the small amounts of zip/MyoII that is maternally loaded and perdures until dorsal closure stages (embryos genetically identical to those assayed in Table 3). During the dorsal closure stage of embryogenesis zip/MyoII localizes in a characteristic ‘bars on a string’ distribution along the leading edge of dorsal-most cells of the lateral epidermis (25,32). Along with F-actin, zip/MyoII forms a contractile supracellular purse string important for dorsal closure completion (25,32).


Figure 3
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  		Figure 3. MYH9-like zip/MyoII mutant heavy chains have a similar subcellular distribution as wild-type GFP-zip/MyoIIWT. Embryos, where the only zygotically expressed zip/MyoII heavy chain is a GFP-zip/MyoII heavy chain, were imaged during the dorsal closure stage of embryogenesis. GFP-zip/MyoIIWT (A), GFP-zip/MyoIIR1171C (B), GFP-zip/MyoIID1430N (C), GFP-zip/MyoIID1847K (D) and GFP-zip/MyoIIR1939X (E). In the dorsal-most cells of the lateral epidermis, each mutant heavy chain localized in a similar distribution as GFP-zip/MyoIIWT throughout closure. This distribution is consistent with endogenous zip/MyoII in these cells (25,32). The scale bar in A is 20 µm and applies to all panels.

 
All GFP-zip/MyoII mutant heavy chains had a subcellular distribution that was indistinguishable from GFP-zip/MyoIIWT in dorsal-most cells (Fig. 3, arrows) showing that all are capable of correct localization. Embryos were observed from just before the onset to the completion of dorsal closure and no differences in the distribution of GFP-zip/MyoII mutant heavy chains in leading edge cells were observed. This suggests that the GFP-zip/MyoII mutant heavy chains act by poisoning endogenous myosin at the site where these proteins usually function.

MYH9-like zip/MyoII heavy chains heterodimerize with endogenous zip/MyoII
We next investigated the ability of mutant heavy chains to dimerize with wild-type heavy chains, a property that has not been determined for any myosin II-related illness. We found that MYH9-like zip/MyoII heavy chains dimerize with endogenous zip/MyoII at near 1:1 stoichiometry (Fig. 4). Using GFP antibodies, we separately immunoprecipitated wild-type and mutant GFP-zip/MyoII heavy chains, from otherwise wild-type larval lysates, under high salt conditions that favor heavy chain dimerization but block the assembly of dimers into filaments. All GFP-zip/MyoII mutant heavy chains were capable of dimerizing with endogenous, wild-type zip/MyoII heavy chain, and each did so with equal stoichiometry within experimental error (Fig. 4). The presence of endogenous zip/MyoII heavy chain in each pull down was specific to dimerization with GFP-zip/MyoII heavy chains as no endogenous zip/MyoII was present in a pull down from a lysate lacking a GFP-zip/MyoII heavy chain (Fig. 4, w1118).


Figure 4
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  		Figure 4. Wild-type and MYH9-like GFP-zip/MyoII heavy chains dimerize with endogenous zip/MyoII. Wild-type and mutant GFP-zip/MyoII heavy chains were expressed ubiquitously in otherwise wild-type larvae. Larval lysates from each, and a control not expressing a GFP-fusion protein (w1118), were prepared under high salt and slightly alkaline conditions (600 mM NaCl, pH 7.8) to ensure only heavy chain dimers were present. GFP antibodies were used to immunoprecipitate the respective GFP-zip/MyoII mutant heavy chains. Samples were separated by SDS–PAGE and immunoblotted with zip/MyoII antibodies. Endogenous, wild-type zip/MyoII heavy chain co-immunoprecipitated with all GFP-zip/MyoII heavy chains demonstrating dimerization occurred. The {alpha}-rabbit IgG blot verifies that GFP antibodies were both added to the lysate and immunoprecipitated. w1118 control demonstrates that under these solution conditions all non-muscle myosin II is soluble and that both GFP-zip/MyoII and endogenous, zip/MyoII are pulled down as a consequence of specific immunoprecipitation. Thus, pull-down of endogenous, wild-type zip/MyoII results from dimerization with GFP-zip/MyoII heavy chains.

 
MYH9-like mutations in zip/MyoII do not affect assembly or coiled-coil structure
Given the in vivo findings that MYH9-like zip/MyoII mutant heavy chains have partial wild-type activity, that they behave dominantly in certain genetic backgrounds and that they dimerize with endogenous zip/MyoII heavy chains, we investigated the effect that MYH9-like rod mutations in zip/MyoII had on the formation of the coiled-coil myosin tail and its self-assembly into higher-order structures. We cloned the same tail fragment (amino acid 1109–1972; Fig. 1A) from each of the MYH9-like zip/MyoII heavy chains into a bacterial protein expression vector, expressed protein in E. coli and purified the protein to homogeneity. The fragments were of similar length to previously studied MYH9 tail fragments (amino acid 1102–1961, 19). We used circular dichroism and electron microscopy of negatively stained specimens and found that {alpha}-helices folded into coiled-coil structures and that those structures could in turn assemble into paracrystals comparable to their wild-type counterparts (data presented in Supplemental Material).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 NOMENCLATURE
 FUNDING
 REFERENCES
 
We have evaluated the efficacy of analyzing the molecular basis of MYH9-related disorders using D. melanogaster as a model system. Under appropriate genetic conditions, substitution of analogous residues in zip/MyoII with any one of four gene lesions (three single amino acid substitutions and one truncation) that cause MYH9-related disorders in humans causes mild dominant phenotypes in D. melanogaster. This suggests that these mutations recapitulate a key feature of the human disorder in this model system. In humans, the dominant phenotypes are so mild that they are frequently discovered only when routine blood smears are performed on patients who present with symptoms due to completely unrelated maladies. In flies, dominant phenotypes are observed in genetic backgrounds that are known to compromise zip/MyoII function. We assert that it would be unlikely to detect mild phenotypes in flies similar to the hematologic manifestations seen in humans—we simply cannot monitor either the structure (anatomy and histology), or the function (physiology) of flies as carefully as we do human patients.

The inability of some embryos to complete development and eclose as adult flies even when the GFP-zip/MyoIIWT construct was expressed (Table 3) is likely the result of the Gal4-UAS expression system and not a result of the biology of GFP-zip/MyoII. We previously showed (25) that expression of transgenes using the Gal4-UAS system often results in the mosaic expression of the transgene, such that a small percentage of cells which should express the transgene fail to do so. These mosaic regions appear to be a random process and occur throughout development. Although non-expressing cells are generally limited to only a few cells in the animal, some animals do exhibit large regions that fail to express GFP-zip/MyoII in target tissues. The absence of an essential protein, like non-muscle myosin II, in even a small population of cells would likely have detrimental effects on development. This may be particularly true during D. melanogaster metamorphosis, when the adult ectoderm is entirely generated from imaginal tissues within the larvae while the existing larval ectoderm is histolysed. Similarly, we also cannot rule out that there are some subtle, inappropriate expression levels in certain cells during development. This could either result from too high, or too low, expression that precludes rescue through the entire life cycle of D. melanogaster to Mendelian expectations.

Our in vitro analysis of tail fragments from the zip/MyoIIMYH9 allele proteins indicates that the mechanism by which these mutant residues influence myosin II function may be at least subtly different in the context of sequences that comprise zip/MyoII versus in the context of sequences that comprise zip/MyoII's human non-muscle myosin IIA homolog (encoded by MYH9). All of the zip/MyoIIMYH9 tail fragments form coiled-coil homodimers as detected by CD spectroscopy and form paracrystals that are comparable to those formed by the parent zip/MyoIIwild type fragment. In contrast, we previously showed that the MYH9 mutant tail fragments either failed to form coiled-coil homodimers and/or failed to assemble into higher order structures like their wild-type counterparts (19). One possibility is that differences in dimerization or assembly of the mutant proteins might be revealed in the future if we performed a more exhaustive exploration of ‘solution’ space in which to perform the analysis of dimerization and assembly. pH, ionic strength, the concentration of divalent cations and the presence of certain anions can all influence the dimerization or assembly of other myosin II heavy chains (33). Alternatively, while the human and D. melanogaster myosin II tails are conserved overall (especially at the amino acid residues studied here, see Fig. 1B–E), there are indeed differences in amino acid sequence between the two heavy chains such that the fragments are 52% identical and 76% similar. It is possible that zip/MyoII may accommodate these point mutations better than MYH9 such that CD spectroscopy and electron microscopy on negatively stained specimens do not have the sensitivity to detect more subtle structural abnormalities. While the function of MYH9-like mutations in zip/MyoII is clearly perturbed in vivo, the effects of these substitutions on dimerization or assembly may simply be too slight to measure in vitro.

The dominant phenotypes that the zip/MyoIIMYH9 alleles display in specific genetic backgrounds indicate that they function like certain other hypomorphic alleles of zip/MyoII and may prove to be excellent bait in screens for genetic interactors (28,30). Such screens have been used to dissect a variety of diverse biological processes, from the form and function of elements of cell structure that provided the first genetic evidence for an in vivo interaction between RhoA and myosin (28,30) to the receptor tyrosine kinase signaling pathways that activate mitogen activated protein kinase (MAP kinase) mediated transcriptional activation (reviewed in 34,35). While one might expect a screen in D. melanogaster to identify some molecules that do not have obvious homologs in humans, conservation of many basic pathways across phylogeny would suggest that a number of interactors would be conserved and provide a facile mechanism to identify putative interactors in vertebrate systems.

The low penetrance of the non-hematologic phenotypes associated with genetic lesions in MYH9 is consistent with these phenotypes requiring a special genetic background for their expression—we hypothesize that the non-hematologic manifestations observed in MYH9-related disorder individuals may only occur in the presence of specific genetic backgrounds, backgrounds not shared by all MYH9 patients.

There is some controversy regarding the mechanism (haploinsufficiency or dominant activity) of how mutations in the MYH9 locus cause the observed human hematologic manifestations. Our findings are not able to formally support, or rule out, either proposed mechanism. However, we note that none of the dominant mutations are due to the kind of mutations that would eliminate myosin function from the perturbed allele (i.e. a molecular null allele due to a nonsense mutation near the 5' end of the open reading frame). We would be surprised if a mild, partial loss of function allele would act in a haploinsufficient fashion.

Compared to humans, most non-muscle myosin II mutant alleles in other organisms result in recessive, loss-of-function gene products that are not dominant. In D. melanogaster, only specifically engineered, truncation alleles of zip/MyoII behave as dominant alleles in an otherwise wild-type background. To date, all sequenced zip/MyoII alleles that are the consequence of a point mutation (e.g. zipEbr, zip6.1, zip2.1) behave as recessive lethal mutations, likely because they resulted from screens designed to recover such recessive alleles. This includes the zip1.3 allele, which encodes a partially stable, truncated heavy chain (the last 74 amino acids are removed, 32). Heterozygous (zip1.3/+) animals are viable with no observed dominant phenotypes while homozygous (zip1.3/ zip1.3) animals arrest similar to null, or near null, alleles (32). In second-site, non-complementation tests zip1.3 did display dominant phenotypes in a RhoA background, but at only low penetrance (RhoAJ3.8+/+zip1.3; 14% of flies were malformed, versus 100% malformed in when the zipEbr allele was used; Susan Halsell unpublished observations). Regardless of the overtly recessive nature of these recessive mutations, they must have some dominant character by the following reasoning: with time, the recessive phenotypes that characterize homozygotes generated from the balanced stocks that are used to propagate these lethal mutations become progressively less expressive, apparently due to the gradual accumulation of suppressors. Out crossing the mutation into a robust, wild-type genetic background reverses the this trend and the recessive phenotype reverts to its original expressivity and penetrance. Finally, we note that we do not know what the homozyogous phenotypes for these dominant MYH9 alleles are in humans (i.e. do these alleles also display a recessive phenotype?). The experimental crosses that are so easy in flies are not feasible in humans.

In vitro studies on mouse and human HMM fragments showed that the dominant, MYH9-like mutations engineered in the motor domain of mouse non-muscle myosin IIC (Myh14; R722C and R730C) and human non-muscle myosin IIB (MYH10; N93K and R709C) cause defects in the enzymatic properties and actin filament sliding compared to wild type (18). In addition, the protein encoded by at least one of these mutations (MYH10; R709C) was able to poison wild-type myosin-mediated actin filament sliding in vitro. Moreover, the in vivo effect (dominant versus recessive) of these mutations is unknown. Takeda et al. (36) and Ma et al. (37) studied the in vivo effects of knocking-in a mutation analogous to the R702C MYH9-related disorder mutation into the mouse non-muscle myosin IIB (Myh10; R709C) locus. Heterozygous knock-in mice displayed no observable phenotypes, indicating that this MYH9-like mutation in Myh10 does not act dominantly. Several defects were found in mice homozygous for the R709C mutation, including abnormal gait, difficulties in maintaining balance and defects in neuronal migration in the brain and face (36). These findings are consistent with our study and show that the R702C MYH9-like mutation in the mouse Myh10 locus behaves recessively. Together, these results indicate that MYH9-related disorder mutations are extraordinarily unique and specific.

Non-muscle myosin II heavy chains, including both MYH9 and zip/MyoII, diverged after non-muscle and muscle myosin II heavy chain isoforms diverged (38). In other words, the MYH9 and zip/MyoII nonmuscle myosin II genes share a common ancestor more recently than MYH9 does with any human muscle myosin II heavy chain. Given our findings, it seems unlikely that any disease-causing mutation in a human muscle myosin II heavy chain would be homologous to any of the MYH9 rod mutations studied here. We aligned all human myosin II heavy chains and found that none of the four MYH9 rod residues studied here have analogous residues in human muscle myosin II heavy chains that are associated with a pathology. Further, we searched the alignment for all identified point mutations in MYH9-related disorder families (13 in total; 6 in the head and 7 in the rod) and found that only one mutation in MYH9 (amino acid 702, described in the Introduction) had an analogous mutation in a different human myosin II heavy chain associated with a pathology (amino acid 726 in MYH14). Interestingly, both genes encode non-muscle heavy chains (MYH9 encodes non-muscle myosin IIA and MYH14 encodes non-muscle myosin IIC).

Together, these findings indicate that each sub class (skeletal muscle, cardiac muscle and non-muscle isoforms) of myosin II heavy chain harbor specific amino acid residues that are particularly susceptible to mutations that result in dominant behavior. Identification of additional disease-causing mutations in different myosin II heavy chain loci will be necessary to better understand the specificity of these mutations. As this study has focused on MYH9 rod mutations, it is not possible to rule out that MYH9-like mutations in the head region (which is more highly conserved) of zip/MyoII may result in dominant heavy chains. Indeed, Bejsovec and Anderson (39) showed that conserved residues in a muscle myosin head from Caenorhabditis elegans would mutate to dominance.

In sum, our results show that amino acid conservation between two non-muscle myosin II heavy chains in humans and flies make D. melanogaster a useful system for applying genetic methods to understanding myosin function in these diverse organisms. Given the broad clinical spectrum and severity of myosin II-related pathologies, this work suggests that rapid genetic screening approaches in D. melanogaster might be used to investigate myosin function in human pathologies.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
NOMENCLATURE
 FUNDING
 REFERENCES
 
Cloning of mutant GFP-zip/MyoII constructs
p[UAS-GFP-zip/MyoII]WT (25) was digested with XbaI to remove a 3450 bp fragment of zip/MyoII that could be easily manipulated. The myosin cDNA sequences were originally recovered (21,40) from a 4–8 h Drosophila embryo library constructed in pNB40 (41). This XbaI fragment includes the following. (i) Amino acids 909 of zip/MyoII (numbering based on the amino acids in the simplest, RC transcript of zip/MyoII) through its translation stop codon (3191 bp). (ii) Two hundred and one base pairs of bona fide 3'-UTR from 3' of the zip/MyoII translation stop through 12 A's of the poly A tail. (iii) Non-palindromic sequences and a Not1 site used for first strand priming, then directional cloning of the zip/MyoII cDNA (23 bp) into pNB40 (41); an additional nucleotide and the EcoR1 site from pNB40; and finally EcoR1 to Xba1 sequences from the pUASt polylinker (31). The complete sequence of p[UAS-GFP-zip/MyoII]WT is provided in Supplemental Material. The 3450 bp XbaI fragment was then ligated into XbaI-digested pBluescript. Site-directed mutagenesis was next performed to make the following MYH9-like rod mutations in the zip/MyoII fragment: R1171C (cgc to tgc), D1430N (gat to aat), D1847K (gac to aag) and R1939X (cgg to tga). Positive clones were sequenced to verify that only the appropriate mutation was present. The mutant 3450 bp fragment was removed from pBluescript by digestion with XbaI, then ligated back into XbaI-digested p[UAS-GFP-zip/MyoII]WT, replacing the wild-type fragment with each mutant and yielding: p[UAS-GFP-zip/MyoII]R1171C, D1430N, D1847K or R1939X. Transgenic flies were generated by standard protocols (42).

Dominant negative and lethal phase rescue analysis using MYH9-like zip/MyoII heavy chains
In vivo dominant negative activity was examined by ubiquitously expressing each MYH9-like zip/MyoII heavy chain in a heterozygous zipper background. P[sqh-Gal4], sp zip1/SM6 virgin females were crossed to males homozygous for a MYH9-like zip/MyoII heavy chain. In the crossing scheme, half of the expected progeny are of the tester genotype and half is wild type with respect to the zip/MyoII background.

Rescue crosses, where the only zygotic zip/MyoII heavy chain transcribed in animals was GFP-zip/MyoII WT, R1171C, D1430N, D1847K or R1939X, were set up as follows: P[sqh-Gal4], sp zip1/SM6 virgin females were crossed to P[UAS-GFP-zip/MyoII]WT, R1171C, D1430N, D1847K or R1939X sp zip2/SM6 males. Fourteen hour embryo collections were performed on standard grape plates and embryos were then collected, dechorionated and selected unambiguously using a fluorescence-dissecting microscope (of all the progeny from this cross, only the rescued embryos have GFP fluorescence). Embryos were placed in a separate bottle to develop at 25°C. Animals were kept at 25°C for the remainder of the experiment and pupae and adult flies were scored. For each individual cross at least 100 embryos of the experimental genotype were selected. Crosses for each genotype were performed in triplicate (therefore, the total number of animals assayed for each genotype was at least 300) and the average and standard deviation was calculated and shown above.

Genetic interaction study using MYH9-like zip/MyoII mutant heavy chains
To examine if MYH9-like zip/MyoII mutant heavy chains exhibited second-site non-complementation characteristics (i.e. could show dominant character in non wild-type genetic backgrounds) virgin P[GFP-zip/MyoII] WT, R1171C, D1430N, D1847K or R1939X sp zip2/SM6 flies were crossed to P[engrailed-Gal4], RhoAE3.10/SM6 male flies. In this cross 33% of eclosing flies were expected to be the tester genotype (those having straight wings; SM6/CyO animals are embryonic or early larval lethal). To verify that effects were specific to the heterozygous RhoA allele, control crosses were set up as follows. Virgin P[GFP-zip/MyoII] WT, R1171C, D1430N, D1847K or R1939X sp zip2 / SM6 females were crossed to P[engrailed-Gal4]/P[engrailed-Gal4] male flies. In this cross 50% of eclosing flies were expected to be the tester genotype.

Brightfield photography of wings and legs
Whole flies were placed directly in 100% ethanol for at least 1 day. Wings and legs were then dissected from these flies and mounted on glass slides using Aquamount (Polysciences Inc., Warrington, PA, USA). Wings and legs were observed by brightfield microscopy using a Zeiss 200M Axiovert microscope (Carl Zeiss, Thornwood, NY, USA) with either 5x (0.15 NA) or 25x (0.8 NA) objectives and images were collected using MetaMorph (Molecular Devices Corporation, Downingtown, PA, USA).

Localization of MYH9-like zip/MyoII mutant heavy chains during dorsal closure
Rescued embryos (as described above) were staged for dorsal closure and mounted for time lapse as previously described (43). A Zeiss 200 M Axiovert inverted microscope with a spinning disc confocal (Perkin Elmer, Boston, MA, USA) was used with a 40x, 0.9 NA multi-immersion lens for imaging.

GFP-zip/MyoIIWT, R1171C, D1430N, D1847K or R1939X heavy chains dimerized with endogenous zip/MyoII heavy chain
To evaluate the ability of each mutant heavy chain to dimerize with wild-type heavy chains we ascertained whether or not anti-GFP antibodies could immunoprecipitate heterodimers from larvae that expressed endogenous, wild-type heavy chain and a GFP-tagged mutant heavy chain as follows: sqh-Gal4 virgin females (which express Gal4 ubiquitously) were crossed to homozygous P[UAS-GFP-zip/MyoII]WT, R1171C, D1430N, D1847K or R1939X and w1118 males (control). Ten 3rd instar larvae resulting from each cross were ground in 1 ml lysis buffer (10 mM Tris-Cl pH 7.8, 20% glycerol, 600 mM NaCl, 1 mM EDTA, 0.2% NP-40, Sigma P8465 protease inhibitors, Sigma-Aldrich, St Louis, MO, USA, and 5 mM ATP), subjected to Dounce homogenization and then spun at 14 000g for 15 min to clear the lysate. Two micro liters of GFP antibody (BD Biosciences 8372-2, Franklin Lakes, NJ, USA) was first incubated with 15 µl of Protein A beads (Zymed Laboratories Inc. 10-1041, Invitrogen, Carlsbad, CA, USA) in 750 µl wash buffer (10 mM HEPES-Cl pH 7.5 and 150 mM NaCl) for 1 h at 25°C. Beads were washed three times in lysis buffer before 900 µl of each cleared larval lysate was added to the beads. Samples were incubated at 4°C for 1 h 30 min. Post-incubation beads were washed five times (2 min and 1 ml per wash) in lysis buffer and SDS-sample buffer was added. Samples were run on a 7% acrylamide SDS–PAGE gel, then proteins were transferred to PVDF membrane and probed for using standard methods (44). {alpha}-zip/MyoII antibody (45) was used at 1:4000 and secondary ({alpha}-rabbit AlexaFluore 680; Molecular Probes Inc., Eugene, OR, USA) was used at 1:5000.


   NOMENCLATURE
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 NOMENCLATURE
FUNDING
 REFERENCES
 
We constructed transgenic animals using standard, P-element-based methods in the D. melanogaster. Thus, all of the animals that include transgenes are genetically marked with mini-white+ and are thus genetically white at their endogenous white loci (present, but not shown in the genotypes listed above). These animals also carry other visible mutations as markers (e.g. sp or Cy) so that we can ascertain individual genotypes unambiguously. Nevertheless, these flies are otherwise wild type, so we designate such flies as being ‘in an otherwise wild-type genetic background’ in order to distinguish them from flies that carry specific mutations at other loci, which do not function as chromosomal markers, and are under experimental investigation (e.g. a RhoA or zip mutant background).


   FUNDING
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 NOMENCLATURE
 FUNDING
REFERENCES
 
Thanks to the University of Puget Sound (UPS) and Dr Scott Weatherwax for supporting the UPS Weatherwax Electron Microscopy Lab. Grant support was from the National Institutes of Health: GM33830 to D.P.K. and GM07184 to J.D.F.


   ACKNOWLEDGEMENTS
 
We would like to thank Amanda Boury for assistance in the site-directed mutagenesis; the Duke University Model Systems Genomics center for generation of transgenic flies and Terry Oas and Preeti Chunga for use of the Duke University CD Spectrometer.

Conflict of Interest statement. None of the authors have any conflict of interest.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 NOMENCLATURE
 FUNDING
 REFERENCES
 

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