A novel class of unconventional myosins from Toxoplasma gondii.

Here, we describe the complete deduced amino acid sequence of three unconventional myosins identified in the protozoan parasite Toxoplasma gondii. Phylogenetic analysis reveals that the three myosins represent a novel, highly-divergent class addition to the myosin superfamily. Toxoplasma gondii myosin-A (TgM-A) is a remarkably small approximately 93 kDa myosin that shows a striking departure from typical myosin heavy chain structure in having a head and tail domain but no discernible neck domain. In other myosins, the neck is defined by one or more IQ motifs that serve as potential light chain binding domains. No IQ motifs are apparent in TgM-A. The tail domain of TgM-A encompasses only 57 amino acid residues and is characterized by its highly basic charge (pI = 10.8). The other two Toxoplasma myosins, TgM-B and TgM-C appear to be the product of differential RNA splicing with TgM-B yielding a protein of approximately 114 kDa and TgM-C a protein of approximately 125 kDa. These two myosins are identical throughout their head domain and neck domain which contains a single IQ motif. TgM-B and C share the proximal 245 residues of their tail domain and then diverge in their tail structure distally. The tails, like that of TgM-A, share no homology to any other myosin tails apart from a highly basic charge. The identification of yet another class of unconventional myosins, including a myosin as novel in structure as the 93 kDa TgM-A, continues to underscore the diversity of this family of molecular motors.

Identification of a divergent actin-related protein in Drosophila.

The actin-related proteins (ARPs) have primary sequence homology to actin, have no homology to other proteins and, unlike the conventional actins, are clearly divergent. We have identified an ARP in Drosophila that has approximately 30% amino acid identity to most actins, making it the most divergent yet reported. It is also quite divergent from all other ARP sequences. When the Drosophila ARP is aligned with actin it contains sequence insertions, as is the case with all other ARPs. The unique location of the insertions, as well as its overall divergence, indicates it may represent a new isotype. Only one gene was detected by hybridization to both genomic DNA and polytene chromosomes; the location of the gene is 13E on the X chromosome. A transcript of 1350 bases was detected at all stages of development. This transcript was relatively abundant during early embryogenesis, decreasing during the later stages of embryogenesis and increasing again in larvae and adults.

Cloning of a secretory gelsolin from Drosophila melanogaster.

Gelsolin is an actin-binding protein with the abilities to sever and cap the barbed end of actin filaments and to promote the assembly of monomeric actin. It has been identified in vertebrates both as a cytoplasmic protein and as a protein secreted into the blood plasma. Here we report the nucleic acid sequence of the full-length complementary DNA for a secretory form of gelsolin from Drosophila. The deduced amino acid sequence of 790 residues (M(r) = 87,669) contains a predicted signal peptide of 20 amino acid residues. Comparison of the Drosophila gelsolin sequence with other members of the gelsolin family of actin-binding proteins reveals the characteristic segmental repeat structure found in this class of proteins. A 42% identity is observed between Drosophila secretory gelsolin and human plasma gelsolin when their primary structures are compared. Northern blots resolve a single 3000 base message in third instar Drosophila larvae, a message that appears to be encoded by a single gene located at 82A, B on the right arm of the third chromosome.

The molecular architecture of an insect midgut brush border cytoskeleton.

The cytoskeletal apparatus of the vertebrate intestinal brush border (BB) has served as a model system for the actin-based cytoskeleton of nonmuscle cells. In this study, we examine the structural organization and molecular architecture of the BB cytoskeleton expressed in the midgut of lepidopteran larvae, Manduca sexta. Electron microscopy of the midgut of the 5th instar larvae revealed enterocytes with an apical BB surface comparable to that in the vertebrate intestine, with both microvillar (MV) and terminal web (TW) domains, the latter defined by a zone of organelle exclusion directly beneath the MV. As reported previously for the larval dragon fly, the MV contain a bundle of actin filaments, as determined by staining with rhodamine phalloidin (Kukulies, J., et al., Protoplasma 121, 157-162 (1984)) and heavy meromyosin decoration (Komnick, H., J. Kukulies, Zoomorphology 107, 241-253 (1987)). Two-dimensional gel analysis revealed the presence of multiple isoelectric variants of actin with the major isoform corresponding to the non-muscle actin isoform II, expressed in Drosophila. Like the vertebrate BB, the Manduca BB can be isolated intact from enterocytes by mechanical shear. Immunochemical analysis of isolated BB fractions or whole homogenates of midgut revealed proteins of appropriate molecular weight immunoreactive with antibodies to the MV core proteins: BB myosin I, villin and fimbrin, and the TW components: spectrin, myosin II and tropomyosin. Immunocytochemical localization of a subset of these proteins at the light microscopic (spectrin) and electron microscopic (actin, villin, spectrin, myosin II, and tropomyosin) level reveals that the molecular architecture of the Manduca BB cytoskeleton is homologous to that found in vertebrates.

Myosin diversity in Apicomplexa.

A polymerase chain reaction (PCR) screen was used to examine the diversity of myosins in 7 Apicomplexan parasites: Toxoplasma gondii, Plasmodium falciparum, Neospora caninum, Eimeria tenella, Sarcocystis muris, Babesia bovis, and Cryptosporidium parvum. Using degenerate PCR primers compatible with the majority of known myosin classes, putative myosin sequences were obtained from all of these species. All of the sequences obtained showed greatest similarity to previously identified apicomplexan myosins, suggesting that the diversity of myosins in these parasites is limited. Myosin classes that are known to be widespread across the phylogenetic spectrum, e.g., the myosins I, II, and V, were not seen in the Apicomplexa. Thus, like the plants, the Apicomplexa may have evolved their own unique cohort of myosins that are responsible for the myosin-driven cellular functions observed in these parasites.

Assembly of the brush border cytoskeleton: changes in the distribution of microvillar core proteins during enterocyte differentiation in adult chicken intestine.

The assembly of the intestinal microvillus cytoskeleton was examined during the differentiation of enterocytes along the crypt-villus axis in adult chicken duodenum using light and electron microscopic immunolocalization techniques. Using antibodies reactive with villin, fimbrin, and the heavy chain (hc) of brush border (BB) myosin I (110K-calmodulin complex) and rhodamine-conjugated phalloidin as a probe for F-actin, we determined that while actin, villin, and fimbrin were all localized apically along the entire axis, BB myosin I (hc) did not assume this localization until the crypt-villus transition zone. In addition to their localization at the BB surface, all four proteins were present at significant levels along the lateral margins of enterocytes along the entire crypt-villus axis, suggesting that these proteins may be involved in the organization and function of the basolateral membrane cytoskeleton as well. The pattern of expression of the microvillar core proteins along the crypt-villus axis in the adult was comparable to that seen in the intestine of the late stage chicken embryo and suggests that a common program for brush border assembly may be used in both modes of enterocyte differentiation.

Structural and compositional analysis of early stages in microvillus assembly in the enterocyte of the chick embryo.

Morphological and immunocytochemical techniques were used to examine the distribution of villin, with respect to actin, during the early events of brush border morphogenesis in the embryonic chicken intestine. Immunolocalization studies indicate that actin and villin exist as a cortical array in the apical domain of embryonic enterocytes at a time when few surface microvilli are visible by scanning and transmission electron microscopic techniques. A population of villin is also localized at the level of the junctional complex. With time, the density of microvilli increases and the cells begin to flatten. In these cells, villin is detected in the newly formed microvilli and also in the subjacent cortex, where microvillar rootlets are beginning to appear. The significance of actin-villin associations in the process of brush border assembly is discussed in the light of the functional properties of villin.

Morphology of ventral epidermis of Rana catesbeiana during metamorphosis.

A detailed morphological examination of the bullfrog tadpole ventral epidermis and changes in structure that occur during metamorphosis has not been done. Knowledge of this is crucial to interpretation of physiological studies such as those dealing with development of transepithelial Na+ transport. Examination of tadpole epidermis with light microscopy reveals the presence of three different cell types: apical, basal, and skein. This epidermal morphology is constant until Taylor and Kollros (Anat. Rec. 94:7-23, 1946) stage 19 when degeneration of apical cells is noted. Stages 20 and 21 are characterized by rapid proliferation of basal cells and development of a true stratum germinativum together with the disappearance of other tadpole cell types. By stage 22, epidermal morphology is similar to that of the adult frog. Studies with the electron microscope reveal that as the proliferation proceeds during metamorphosis, the skein cells, at stage 20, differentiate to form the apical border of the skin. The development of the adult frog cell phenotype appears to mimic the cellular differentiation that occurs in the adult epidermis with the cells first developing into progranular cells in the intermediate stratum of the skin and then progressing to granular cells in the outermost living cell layer. The granular cells then undergo cornification to form the stratum corneum. Mitochondria rich cells are not seen in the developing epidermis until stage 21. These observations, when considered with previous results from Na+ transport studies (Hillyard et al.: Biochim. Biophys. Acta 692:455-461, 1982), suggest that both the physiological differentiation and morphological differentiation are simultaneous events.

Characterization of myosin-A and myosin-C: two class XIV unconventional myosins from Toxoplasma gondii.

Two class XIV unconventional myosins from Toxoplasma gondii, Myosin-A (TgM-A) and Myosin-C (TgM-C), were characterized in terms of their biochemical properties and their expression in quiescent and motile stages of the parasite life cycle. In cell fractionation studies, both myosins partitioned with the major organelle/cell membrane fraction, and extraction studies indicated that both were tightly associated with membrane domains as detergent was necessary for their solubilization. In addition, both TgM-A and TgM-C demonstrated a hallmark feature of myosins in their ability to bind actin in the absence but not the presence of ATP. In parasites residing within the host cell parasitophorous vacuole, TgM-A was detected by immunofluorescence microscopy as a bright spot near the apical pole of the parasite. This pattern underwent a subtle change as the parasites became motile, with TgM-A then localizing more intimately with the parasite cell membrane domain in apically disposed spots or patches, consistent with the role of this myosin in gliding motility. TgM-C showed a distinct localization to the juxtanuclear region towards the apical pole of the parasite, consistent with an association with the Golgi apparatus.

Characterization of myosin-IA and myosin-IB, two unconventional myosins associated with the Drosophila brush border cytoskeleton.

The expression patterns of myosin-IA (MIA) and myosin-IB (MIB), two novel unconventional myosins from Drosophila melanogaster, have been characterized through immunoblot analysis and immunocytochemistry of embryos, larvae, and adults. The appearance and distribution of both proteins during embryogenesis is correlated with the formation of a brush border within the alimentary canal as documented at the ultrastructural level. MIA and MIB, both found predominantly at the basolateral domain of immature enterocytes, exhibit increased expression at the apical domain of differentiated enterocytes co-incident with microvillus assembly. Colocalization of MIA and MIB to larval and adult gut by confocal microscopy demonstrates distinct but overlapping subcellular distributions of these two proteins. In the larval brush border, MIA is enriched in the subapical terminal web domain whereas MIB is found predominantly in the apical microvillar domain. In the adult gut, MIA and MIB both exhibit a microvillar component as MIA attains a more apical position in addition to its previous terminal web locale. MIB is also found in egg chambers at both the basolateral and apical surfaces of the somatic follicle cells during oogenesis. MIA and MIB both demonstrate ATP-dependent extraction from the larval brush border cytoskeleton and exogenous F-actin, biochemical properties characteristic of functional myosins-I.

Multiple unconventional myosin domains of the intestinal brush border cytoskeleton.

Representatives of class V and class VI unconventional myosins are identified as components of the intestinal brush border cytoskeleton. With brush border myosin-I and myosin-II, this brings to four the number of myosin classes associated with this one subcellular domain and represents the first characterization of four classes of myosins expressed in a single metazoan cell type. The distribution and cytoskeletal association of each myosin is distinct as assessed by both biochemical fractionation and immunofluorescence localization. Myosin-VI exists in both the microvillus and terminal web although the terminal web is the predominant site of concentration. Myosin-V is present in the terminal web and, most notably, at the distal ends of the microvilli, thus becoming the first actin-binding protein to be localized to this domain as assessed by both immunohistochemical and biochemical methods. In the undifferentiated enterocytes of the intestinal crypts, myosin-VI is expressed but not yet localized to the brush border, in contrast to myosin-V, which does demonstrate an apical distribution in these cells. An assessment of myosin abundance indicates that while myosin-II is the most abundant in the cell and in the brush border, brush border myosin-I is only slightly less abundant in contrast to myosins-V and -VI, both of which are two orders of magnitude less abundant than the others. Extraction studies indicate that of these four myosins, myosin-V is the most tightly associated with the brush border membrane, as detergent, in addition to ATP, is required for efficient solubilization.

ZO-1 and cingulin: tight junction proteins with distinct identities and localizations.

The relative localization of ZO-1 and cingulin, the only two known components of the tight junction, was compared in Madin-Darby canine kidney (MDCK) cells, chicken small intestine, rat kidney distal convoluted tubule, and a hepatoma cell line. Immunoblot analysis demonstrated that cingulin and ZO-1 are immunologically unrelated and that, in the colon, cingulin is a single polypeptide with a molecular mass of 140 kDa. Immunofluorescent localization of cingulin and ZO-1 in confluent monolayers of MDCK cells showed identical staining patterns. However, subconfluent MDCK cells showed distinct localizations of the two proteins. Both cingulin and ZO-1 were found at the plasma membrane only at areas of cell-cell contact, but cingulin was diffusely distributed within the cytoplasm, whereas ZO-1 showed a more clustered internal arrangement. Cingulin and ZO-1 were identically localized at the plasma membrane of hepatoma tissue culture (HTC) cells at sites of cell-cell contact. In chicken intestine examined at the ultrastructural level, immunogold particles associated with cingulin were found approximately three times farther from the junctional membrane than those affiliated with ZO-1.

Expression in cochlea and retina of myosin VIIa, the gene product defective in Usher syndrome type 1B.

Myosin VIIa is a newly identified member of the myosin superfamily of actin-based motors. Recently, the myosin VIIa gene was identified as the gene defective in shaker-1, a recessive deafness in mice [Gibson, F., Walsh, J., Mburu, P., Varela, A., Brown, K.A., Antonio, M., Beisel, K.W., Steel, K.P. & Brown, S.D.M. (1995) Nature (London) 374, 62-64], and in human Usher syndrome type 1B, an inherited disease characterized by congenital deafness, vestibular dysfunction, and retinitis pigmentosa [Weil, D., Blanchard, S., Kaplan, J., Guilford, P., Gibson, F., Walsh, J., Mburu, P., Varela, A., Levilliers, J., Weston, M.D., Kelley, P.M., Kimberling, W.J., Wagenaar, M., Levi-Acobas, F., Larget-Piet, D., Munnich, A., Steel, K.P., Brown, S.D.M. & Petit, C. (1995) Nature (London) 374, 60-61]. To understand the normal function of myosin VIIa and how it could cause these disease phenotypes when defective, we generated antibodies specific to the tail portion of this unconventional myosin. We found that myosin VIIa was expressed in cochlea, retina, testis, lung, and kidney. In cochlea, myosin VIIa expression was restricted to the inner and outer hair cells, where it was found in the apical stereocilia as well as the cytoplasm. In the eye, myosin VIIa was expressed by the retinal pigmented epithelial cells, where it was enriched within the apical actin-rich domain of this cell type. The cell-specific localization of myosin VIIa suggests that the blindness and deafness associated with Usher syndrome is due to lack of proper myosin VIIa function within the cochlear hair cells and the retinal pigmented epithelial cells.

Centrin is a conserved protein that forms diverse associations with centrioles and MTOCs in Naegleria and other organisms.

Centrin, a approximately or equal to 20 kDa calcium-binding protein also known as caltractin, is a component of centrosome-associated algal flagellar roots capable of calcium-mediated contraction, and is also found in the centrosomes of vertebrate cells. Our analysis of a centrin gene from a protist, the amoeboflagellate Naegleria gruberi, reveals conserved features that distinguish centrins from calmodulin. Antibodies to bacterially expressed Naegleria centrin, which also recognize yeast Cdc31p, were employed to localize centrin immunoreactivity in selected organisms possessing specialized microtubule-organizing centers (MTOCs) or accessory structures. There is a striking morphological diversity of such structures. In the simplest associations, as found in Naegleria flagellates and vertebrates tracheal epithelium, centrin is intimately associated with the cylinder of the basal bodies. In cells with unfocused mitotic spindles, Naegleria amoebae and onion root tips, no localization of centrin was detected. In Dictyostelium discoideum and Saccharomyces cerevisiae, which lack centrioles, centrin immunoreactivity was observed as punctate cytoplasmic bodies but not associated with spindle pole MTOCs. In Paramecium multimicronucleatum, centrin immunoreactivity is localized to the infraciliary lattice, previously shown to exhibit calcium-mediated contraction. In Vorticella microstoma, known for the calcium-induced rapid contraction of its stalk, centrin immunoreactivity is localized to the contractile spasmoneme and myonemes. Similar antigens from Paramecium and Vorticella are detected by anti-centrin and anti-spasmin. The pattern of localization of centrin immunoreactivity supports the conjecture that a contractile system involving centrin, initially associated with centriolar structures, was recruited during evolution to build specialized organelles in different organisms and cell types.