Identification and overlapping expression of multiple unconventional myosin genes in vertebrate cell types.

Myosin divers In the human epi cell line Caco-2Ba, the porcine pihal Cell lineLLC PK1 (CL-4)9 human peripheral blood leukocytes, and human liver was analyzed. PCR amplification yielded 8-11 putative myouins (depending on the cDNA source) representing six distinct myosin dasses. Analysis of clones obtained by hybridization screening demonstrated that the inal PCR products correspond to bona fide myosins, based on the presence of sequences highly conserved in other myosins. RNase protection analysis confirmed mRNA expression of 11 myosins in Caco-2B&3 cells. Immunoblot analysis showed that at least 6 myosin immunogens are expressed in Caco-2BB. cells. The results reveal the existence of at least 11 unconventional human myosin genes, most of which are expes in an overilapping fashion in different cell types. The abundance ofmyosin suggests that the myosin I vs. myosin II paradin is inadequate to explain actin-based cellular motility. Eukaryotes display a broad array of motility phenomena, ranging from muscle-driven movement to translocation of intracellular organelles. The mechanisms underlying these phenomena are not entirely clear, but it is generally thought that "molecular motors," such as kinesins, dyneins, and myosins, act at cytoskeletal-cytoskeletal and cytoskeletalmembrane interfaces to power organism and cellular movements (1-4). These motors are represented by multiple members in eukaryotes (4, 5), each of which is presumed to play one or more roles during the life of the organism. For example, the fruit fly is known to have at least 6 (6) and possibly as many as 30 (7) kinesins. A unicellular organism, Dictyostelium discoideum, has a minimum offive myosins (8, 9) and probably more than nine (10, 47). Thus, within an organism, multiple diverse molecular motors are the rule. For myosins, phylogenetic analyses have revealed the existence of eight classes (11-13), termed either conventional (filament-forming myosins II) or unconventional (apparently nonfilamentous myosins I and III-VIII) (14). Although evidence indicates that many ofthe unconventional myosin classes will be represented in a given organism (5, 15), to our knowledge, in no case has the number of myosins expressed in a metazoan cell type been determined. Consequently, studies concerning actin-based motility in metazoan cells are complicated by limited information on cellular myosin diversity. We therefore analyzed myosin diversity in two epithelial cell lines, Caco2BBe (16) and LLC-PK1 [CL-4] (17), and in human leukocytes and liver.1 Surprisingly, these cell types express six myosin classes, most of which have at least two representatives. The results challenge the standard "myosin I vs. myosin II" paradigm for actin-based movement in the typical eukaryotic cell. MATERIALS AND METHODS PCR Amplification of Myosins, Sequencing, and Library Screening. Caco-2BBe cells were cultured as described (16). LLC-PK1 (CL-4) cells were obtained from C. Slayman (Yale University), cultured in minimum essential medium, a modification (GIBCO/BRL)/10% (vol/vol) fetal calf serum/2% (wt/vol) glutamine (Sigma) and penicillin 100 (units/ml)/streptomycin (100 t.g/ml)/fungizone (0.25 pug/ ml) (JRH Biosciences, Lenexa, KS), and used at 4 days after confluence. Peripheral blood leukocytes were purified from whole blood of human volunteers via density gradient cell separation (18). RNA was isolated by guanidinium lysis followed by a CsCl gradient (19). cDNAs were generated using reverse transcriptase (New England Biolabs). For PCR of liver cDNAs, a AZAP cDNA library (Stratagene) prepared from normal human liver (a gift from James Mustafa Anderson, Yale University) was employed. Myosins were amplified from cDNAs using standard PCR techniques (20). Two degenerate myosin primers, "ATP-3" (upstream) [GGIGA(G/A)(A/T)(G/C)IGGIGCIGGXAA(G/A)AC] and "EAF-A" (downstream) [GT(C/T)TTIGC(G/A)AAIGC(C/T)TC], were designed based on the myosin consensus sequence (2). ATP-3 corresponds to the amino acid sequence GESGAGKT, a sequence found in all myosins (e.g., aa 163-170 in avian myosin V; ref. 21), and EAF-A corresponds to EAFGNAKT, a sequence found in most myosins. After amplification, products were separated on NuSieve (FMC) agarose gels, excised from the gel, and reamplified by PCR. After the second amplification, products were separated on agarose gels and purified on DEAE-cellulose paper. Purified products were ligated into T-modified Bluescript vector (22) and transformed into competent bacteria (XL1-Blue, Stratagene). Plasmid minipreps (23) were performed and insert presence was confirmed by restriction analysis. Inserts were sequenced with a kit (United States Biochemical). Larger clones were obtained by hybridization screening of the human liver library with a subset offour ofthe subcloned PCR products. Plasmid inserts were labeled with 32P by using random priming (Boehringer Mannheim). Positive plaques were isolated and converted to phagemid by in vivo excision. Expressio Analysis. RNase protection probes were generated using subcloned PCR products. Antisense [32P]UTP and rCTP-labeled RNA were transcribed from plasmids using T3 or T7 RNA polymerase. Probes were hybridized with 20 tig of total Caco-2BB. RNA, digested with RNases A and T1 (19), and separated on 8% polyacrylamide gels, using endlabeled A DNA digested with Hinfl as size markers. Because Abbreviations: HuncM, human unconventional myosin; HConM, human conventional myosin; PuncM, porcine unconventional myosin; PConM, porcine conventional myosin. 'The sequences reported in this paper have been deposited in the GenBank data base (accession nos. L29137-L29149 for human unconventional myosins and L29128-L29136 for porcine unconventional myosins). 6549 The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. Proc. Nail. Acad. Sci. USA 91 (1994) probes were derived from PCR products containing inosine residues at their 5' and 3' ends and because primer matching need not be exact for amplification, two complications must be considered. (i) The probes were heterogenous at their ends, thus, protected products often resolved as several bands on autoradiographs. (ii) It was not possible to predict the exact size of protected products; rather, size predictions were limited to the range, with the region between the primers defining the lower limit (90-120 bp) and this region plus the primers defining the upper limit (136-166 bp). Nevertheless, RNase protection using degenerate PCR products provides a reproducible and sensitive means to assess myosin RNA expression (24). Flasks containing Caco-2BBe cells were rinsed with TBS (150 mM NaCI/50 mM Tris'HCI, pH 7.4), scraped into a polypropylene tube, pelleted, and homogenized in SDS sample buffer. Samples were separated on 5-15% polyacrylamide gels, electroblotted to nitrocellulose (16), and probed with anti-human platelet myosin II antibodies (Biomedical Technologies, Stoughton, MA), affinity-purified anti-chicken A. Human Myosin-like PCR Products