Brush Border myosin-I Research Articles

Myosin-I isozymes in neonatal rodent auditory and vestibular epithelia.

Myosin isozymes are essential for hair cells, the sensory cells of the inner ear. Because a myosin-I subfamily member may mediate adaptation of mechanoelectrical transduction, we examined expression of all eight myosin-I isozymes in rodent auditory and vestibular epithelia. Using RT-PCR, we found prominent expression of three isozymes, Myo1b (also known as myosin-Ia or myr 1). Myo1c (myosin-Ib or myr 2). and Myo1e (myr 3). By contrast, Myo1a (brush-border myosin-I), Myo1d (myosin lg or myr 4). Myo1f, Myo1g, and Myo1h were less readily amplified. Because sequence analysis demonstrated that the RT-PCR products encoded the appropriate isozymes, this represents the first demonstration of expression of all eight mouse myosin-I genes. Using immunocytochemistry with isozyme-selective antibodies, we found that Myo1b was located at apical surfaces of supporting cells that surround hair cells in auditory epithelia of postnatal rats. In vestibular epithelia, Myo1b was present in a ring within the apical pole of the hair cell. In both cases, expression was prominent only immediately after birth. Myo1e was found in hair cells of the auditory and vestibular epithelia; this isozyme was enriched in the cuticular plate, the actin meshwork that anchors the stereocilia. Myo1c was found in hair-cell stereocilia, concentrated towards their tips; we confirmed this localization by using adenovirus vectors to direct expression of a GFP-Myo1c tail fusion protein; this fusion protein localized to plasma membranes, often concentrating at stereociliary tips. Myo1c therefore remains the myosin isozyme best localized to carry out transducer adaptation.

Brush border myosin I (BBMI): a basally localized transcript in human jejunal enterocytes.

To extend our recent observation that villin mRNA, encoding an apical microvillous protein, is dichotomously localized in the basal region of human enterocytes, we examined the localization of mRNAs for brush border myosin I (BBMI) and intestinal fimbrin (I-fim). In situ hybridization indicated that BBMI mRNA localized to the basal region of human enterocytes, whereas the mRNA for I-fim distributed diffusely. To facilitate study of potential mechanisms of mRNA targeting, we cloned a full-length cDNA for BBMI including its 5'- and 3'-untranslated regions (UTRs). This cDNA shares 86% sequence identity with bovine BBMI and 85% with rat BBMI. Sequence analysis revealed no obvious similarity between the 3'-UTRs of BBMI and villin. This study provides evidence of novel sorting pathways for intestinal microvillous cytoskeletal proteins.

Cloning and characterization of mouse brush border myosin-I in adult and embryonic intestine

Brush border myosin-I is a class I myosin with calmodulin light chains that has been identified in several vertebrate species. In chicken, it is exclusively expressed in intestinal epithelial cells where it forms spirally arrayed bridges that tether the microvillar actin bundle to the membrane. To facilitate future knockout strategies, we have isolated mouse brush border myosin-I cDNA and genomic clones. The deduced primary structure of mouse brush border myosin-I is homologous to other known brush border myosins-I. Northern blot, immunoblot, and immunolocalization studies indicate that the intestine-specific and subcellular localization profile of mouse brush border myosin-I are comparable to that determined for other brush border myosins-I. Northern analysis during embryogenesis revealed a 3.9-kb transcript first detected in 15-day embryos. This is in marked contrast to chicken, where brush border myosin-I expression begins early in embryogenesis. In situ localization in 17-day embryos indicated that RNA expression is restricted to the intestine. Protein expression is first detected in 16-day embryos with decreasing levels observed in a proximal to distal fashion. Immunolocalization in embryonic intestine revealed that brush border myosin-I is evenly distributed on both apical and basolateral membrane domains. There is also pronounced localization to a supranuclear region, presumably the Golgi apparatus. This suggests that brush border myosin-I may be targeted to the plasma membrane on Golgi-derived vesicles rather than by direct targeting to microvillar actin cores.

Structure and Action of Molecular Tracks and Motors

Abstract Molecular motors belonging to the myosin and kinesin superfamilies utilize ATP to move along their respective F-actin and microtubule tracks. The track-motor complexes have not been amenable to crystallization, so x-ray crystallographic investigations have focused on structure determinations of the individual proteins. Although providing detailed descriptions of the structure of each protein, this approach cannot reveal the geometry of interaction of the proteins nor the conformational changes which occur during the mechanochemical cycle. To obtain this information, we use cryoelectron microscopy and image analysis to calculate three dimensional maps of the track-motor complexes at moderate resolution (15-30A) and combine these data with the high resolution x-ray structures to provide near-atomic models of the working assemblies. We have so far built models of the rigor (nucleotide-free) and ADP actomyosin complexes. In smooth muscle myosin II (a collaboration with H.L. Sweeney, U. Perm.) and brush border myosin I (BBMI), the motor domain of the myosin head is similar in both biochemical states.

DISTINCT mRNA DISTRIBUTION PATTERNS IN HUMAN JEJUNAL ENTEROCYTES.

70 Although intracellular sorting of mRNAs has been demonstrated in several cellular systems, including oocytes, embryos, neurons, fibroblasts, and absorptive enterocytes, its importance in enterocyte differentiation is poorly understood. Previous studies from our laboratory have demonstrated that mRNAs are asymmetrically distributed in human intestinal epithelial cells, but in general co-localized with their encoded proteins. Most mRNA sorting studies indicate that cis-acting sequences responsible for mRNA localization are located in the 3′-untranslated region (3′UTR). The aims of this study were to characterize in human enterocytes the mRNA localization patterns of genes with distinctly different functions: villin, brush border myosin I (BBMI), Na+-dependent glucose co-transporter (SGLT1), and liver fatty acid binding protein (L-FABP), and to characterize the protein(s) that may be involved in the mRNA sorting process. Methods: mRNA distribution in formalin fixed human jejunal tissue sections was determined byin situ hybridization at high stringency using digoxigenin-labeled RNA probes. Gel-shift assays were used to study RNA-protein interactions in the 3′UTR. Results: The mRNAs for villin and BBMI, well-characterized apical microvillus cytoskeletal proteins, were unexpectedly sorted to the basal region of enterocytes, a different intracellular location than their encoded proteins. The mRNA for human SGLT1 was sorted to the apical region, a location near its encoded protein; and the mRNA for human L-FABP was distributed diffusely in the cytoplasm as is its encoded protein. Gel-shift assays indicated that a cytoplasmic protein in Caco-2 cells binds to the villin 3′UTR, suggesting that this region contains a protein-binding motif. Conclusions: This study not only demonstrates distinct mRNA sorting patterns in human absorptive enterocytes for villin, BBMI, SGLT1, and L-FABP, but also reveals for the first time that a dichotomy may exist between the distribution pattern of mRNA and that of its sorted protein. The observations further offer the opportunity to identify the mechanisms by which mRNAs are separately localized to apical and basal domains in absorptive enterocytes.

Three-dimensional structure of Acanthamoeba castellanii myosin-IB (MIB) determined by cryoelectron microscopy of decorated actin filaments.

The Acanthamoeba castellanii myosin-Is were the first unconventional myosins to be discovered, and the myosin-I class has since been found to be one of the more diverse and abundant classes of the myosin superfamily. We used two-dimensional (2D) crystallization on phospholipid monolayers and negative stain electron microscopy to calculate a projection map of a "classical" myosin-I, Acanthamoeba myosin-IB (MIB), at approximately 18 A resolution. Interpretation of the projection map suggests that the MIB molecules sit upright on the membrane. We also used cryoelectron microscopy and helical image analysis to determine the three-dimensional structure of actin filaments decorated with unphosphorylated (inactive) MIB. The catalytic domain is similar to that of other myosins, whereas the large carboxy-terminal tail domain differs greatly from brush border myosin-I (BBM-I), another member of the myosin-I class. These differences may be relevant to the distinct cellular functions of these two types of myosin-I. The catalytic domain of MIB also attaches to F-actin at a significantly different angle, approximately 10 degrees, than BBM-I. Finally, there is evidence that the tails of adjacent MIB molecules interact in both the 2D crystal and in the decorated actin filaments.

Characterization of human brush border myosin I (BBMI) cDNA and basal localization of its mRNA in jejunal enterocytes

Characterization of human brush border myosin I (BBMI) cDNA and basal localization of its mRNA in jejunal enterocytes

Human brush border myosin-I and myosin-Ic expression in human intestine and Caco-2BBe cells.

The human intestinal cell line, Caco-2BBe, has been established as an excellent model system for analysis of the enterocyte cytoskeleton including that of the actin rich apical brush border. To facilitate its use for functional analysis of a major component of the brush border, brush border myosin-I, human cDNAs encoding the heavy chain of this class I myosin were isolated and sequenced. The identity of this myosin as human brush border myosin-I was verified based on similarity with other vertebrate sequences, as well as its expression profile at both the RNA and protein levels. Localization of the protein in human intestine along the crypt-villus axis closely resembles that previously determined for brush border myosin-I in chicken, and is quite distinct from that of myosin-Ic, another myosin-I expressed in human intestine and Caco-2BBe cells. In immature cells of the crypt, brush border myosin-I staining is low, and there is significant cytosolic and basolateral localization, while villus cells stain much more intensely, and the protein is primarily localized to the brush border. Localization of myosin-Ic is essentially the inverse of brush border myosin-I in that crypt cells exhibit higher levels of staining, while villus cells have very low levels of myosin-Ic. The expression of both myosins-I was also examined during cell-contact induced differentiation of Caco-2BBe cells where expression and changes in localization closely resemble those that accompany differentiation of enterocyte in vivo.

Human brush border myosin‐I and myosin‐Ic expression in human intestine and Caco‐2BBe cells

The human intestinal cell line, Caco-2BBe, has been established as an excellent model system for analysis of the enterocyte cytoskeleton including that of the actin rich apical brush border. To facilitate its use for functional analysis of a major component of the brush border, brush border myosin-I, human cDNAs encoding the heavy chain of this class I myosin were isolated and sequenced. The identity of this myosin as human brush border myosin-I was verified based on similarity with other vertebrate sequences, as well as its expression profile at both the RNA and protein levels. Localization of the protein in human intestine along the crypt-villus axis closely resembles that previously determined for brush border myosin-I in chicken, and is quite distinct from that of myosin-Ic, another myosin-I expressed in human intestine and Caco-2BBe cells. In immature cells of the crypt, brush border myosin-I staining is low, and there is significant cytosolic and basolateral localization, while villus cells stain much more intensely, and the protein is primarily localized to the brush border. Localization of myosin-Ic is essentially the inverse of brush border myosin-I in that crypt cells exhibit higher levels of staining, while villus cells have very low levels of myosin-Ic. The expression of both myosins-I was also examined during cell-contact induced differentiation of Caco-2BBe cells where expression and changes in localization closely resemble those that accompany differentiation of enterocyte in vivo. Cell Motil. Cytoskeleton 41:308–324, 1998. © 1998 Wiley-Liss, Inc.

Kinetic characterization of brush border myosin-I ATPase.

Brush border myosin-I (BBM-I) is a single-headed unconventional myosin found in the microvilli of intestinal epithelial cells. We used stopped-flow kinetic analysis to measure the rate and equilibrium constants for several steps in the BBM-I ATPase cycle. We determined the rates for ATP binding to BBM-I and brush border actomyosin-I (actoBBM-I), the rate of actoBBM-I dissociation by ATP, and the rates for the steps in ADP dissociation from actoBBM-I. The rate and equilibrium constants for several of the steps in the actoBBM-I ATPase are significantly different from those of other members of the myosin superfamily. Most notably, dissociation of the actoBBM-I complex by ATP and release of ADP from actoBBM-I are both very slow. The slow rates of these steps may play a role in lengthening the time spent in force-generating states and in limiting the maximal rate of BBM-I motility. In addition, release of ADP from the actoBBM-I complex occurs in at least two steps. This study provides evidence for a member of the myosin superfamily with markedly divergent kinetic behavior.

Brush border myosin-I structure and ADP-dependent conformational changes revealed by cryoelectron microscopy and image analysis.

Brush border myosin-I (BBM-I) is a single-headed myosin found in the microvilli of intestinal epithelial cells, where it forms lateral bridges connecting the core bundle of actin filaments to the plasma membrane. Extending previous observations (Jontes, J.D., E.M. Wilson-Kubalek, and R.A. Milligan. 1995. Nature [Lond.]. 378:751-753), we have used cryoelectron microscopy and helical image analysis to generate three-dimensional (3D) maps of actin filaments decorated with BBM-I in both the presence and absence of 1 mM MgADP. In the improved 3D maps, we are able to see the entire light chain-binding domain, containing density for all three calmodulin light chains. This has enabled us to model a high resolution structure of BBM-I using the crystal structures of the chicken skeletal muscle myosin catalytic domain and essential light chain. Thus, we are able to directly measure the full magnitude of the ADP-dependent tail swing. The approximately 31 degrees swing corresponds to approximately 63 A at the end of the rigid light chain-binding domain. Comparison of the behavior of BBM-I with skeletal and smooth muscle subfragments-1 suggests that there are substantial differences in the structure and energetics of the biochemical transitions in the actomyosin ATPase cycle.

Association of a Brush Border Myosin I-Like Protein with Vesicular Organelles of the Secretory Pathway in Chicken Enterocytes

Brush border myosin I (BBMI) was first identified as a major constituent of the microvillus cytoskeleton, where it links the actin filaments of the microvillus core to the overlying plasma membrane. Recently, however, BBMI was also found on isolated Golgi membranes. This observation suggests that cytoplasmic BBMI might act as a molecular motor for apically targeted secretory vesicles.In an earlier study from this laboratory a 105-110 kD protein was isolated from the chicken small intestine, which displayed several characteristics of the BBMI. Using an antibody against this BBMI- like protein (MLP), the protein was detected in the apical brush border of the enterocytes. The limited resolution of the light microscopic immunohistochemical techniques used in that investigation, however, did not provide information about the intracellular distribution of MLP in the enterocytes.In this study the intracellular localization of MLP was characterized using post-embedding colloidal gold immunocytochemistry. Pieces of the jejunum from 6-week-old male White Leghorn chickens were fixed and embedded in LR Gold resin.

Conformational changes due to calcium-induced calmodulin dissociation in brush border MyosinI-decorated F-actin revealed by cryoelectron microscopy and image analysis

Brush border myosin I (BBMI) is a single-headed molecular motor. Its catalytic domain exhibits extensive sequence homology to the catalytic domain of myosin II, while its tail lacks the coiled-coil nature of myosin II. The BBMI tail domain contains at least three IQ motifs and binds calmodulin. Addition of calcium removes one of these calmodulin light chains, with effects on ATPase activity and motility in in vitro assays. Using the techniques of cryoelectron microscopy and helical image analysis we have calculated three-dimensional (3D) maps of BBMI-decorated actin filaments prepared in the presence and absence of calcium. The 3D maps describe a BBMI catalytic domain that is strikingly similar to the catalytic domain of myosin II subfragment 1 (S1), with the exception of a short amino-terminal region of the heavy chain, which is absent from BBMI. The tail domains of BBMI and S1 are highly divergent in structure, continuing on from their respective motor domains with very different geometries. Addition of calcium to BBMI, and the concomitant loss of a calmodulin light chain, results in an extensive reorganization of mass in the tail domain.

Three-dimensional structure of brush border myosin-I at ∼20 å resolution by electron microscopy and image analysis

Brush Border Myosin-I (BBMI) is a single-headed, unconventional myosin found in the microvilli of intestinal epithelial cells where it forms lateral bridges between the core bundle of actin filaments and the plasma membrane of the microvillus. A three-dimensional (3D) reconstruction of BBMI was made from images of negatively stained, two-dimensional (2D) crystals grown on lipid monolayers formed from mixtures of phosphatidylserine and phosphatidylcholine. The resolution of the 3D map extends to ∼20 Å and allows identification of all of the major structural domains of BBMI. The BBMI molecule is composed of three domains: a globular motor domain, a light-chain-binding domain and a lipid-binding domain. In our map, the putative motor domain is connected to an extended density, which we believe to be the light-chain-binding domain. This long, narrow region has three distinct bends, which may delineate the bound calmodulin light chains. Following the last calmodulin there is density which extends for a short distance across the lipid surface and is presumably the carboxy-terminal lipid-binding domain.

Two-Dimensional Crystallization of Brush Border Myosin I

Brush border myosin-I (BBMI) is a single-headed unconventional myosin found in the microvilli of intestinal epithelial cells, where it links the core bundle of actin filaments to the plasma membrane. An association of BBMI with anionic phospholipids has been shown to be mediated by a carboxy-terminal domain which is rich in basic amino acids. We have exploited this natural affinity of BBMI for negatively charged lipids to form two-dimensional (2D) crystals of this protein which are suitable for structural analysis by electron crystallographic techniques. The 2D crystals which we have obtained belong to one of two space groups, p22121or p2. We present here projection maps calculated from images of negatively stained crystals for each of these crystal types to a resolution of 20 Å and show that the asymmetric unit is the same in both crystal types.

Brush border myosin-I truncated in the motor domain impairs the distribution and the function of endocytic compartments in an hepatoma cell line.

Myosins I, a ubiquitous monomeric class of myosins that exhibits actin-based motor properties, are associated with plasma and/or vesicular membranes and have been suggested as players for trafficking events between cell surface and intracellular membranous structures. To investigate the function of myosins 1, we have transfected a mouse hepatoma cell line (BWTG3) with cDNAs encoding the chicken brush border myosin-I (BBMI) and two variants truncated in the motor domain. One variant is deleted of the first 446 amino acids and thereby lacks the ATP binding site, whereas the other is deleted of the entire motor domain and lacks the ATP and actin binding sites. We have observed (i) that significant amounts of the truncated variants are recovered with membrane fractions after cell fractionation, (ii) that they codistribute with a compartment containing alpha2-macroglobulin internalized for 30 min as determined by fluorescent microscopy, (iii) that the production of BBMI-truncated variants impairs the distribution of the acidic compartment and ligands internalized for 30 min, and (iv) that the production of the truncated variant containing the actin binding site decreases the rate of alpha2-macroglobulin degradation whereas the production of the variant lacking the ATP binding site and the actin binding site increases the rate of a2-macroglobulin degradation. These observations indicate that the two truncated variants have a dominant negative effect on the distribution and the function of the endocytic compartments. We propose that an unidentified myosin-I might contribute to the distribution of endocytic compartments in a juxtanuclear position and/or to the regulation of the delivery of ligands to the degradative compartment in BWTG3 cells.

Enzymatic Characterization and Functional Domain Mapping of Brain Myosin-V

The actin binding and ATPase properties, as well as the functional domain structure of chick brain myosin-V, a two-headed, unconventional myosin, is reported here. Compared to conventional myosin from skeletal muscle, brain myosin-V exhibits low K-EDTA- and Ca-ATPase activities (1.8 and 0.8 ATP/s per head). The physiologically relevant Mg-ATPase is also low (approximately 0.3 ATP/s), unless activated by the presence of both F-actin and Ca2+ (Vmax of 27 ATP/s). Ca2+ stimulates the actin-activated Mg-ATPase over a narrow concentration range between 1 and 3 microM. In the presence of saturating Ca2+ and 75 mM KCl, surprisingly low concentrations of F-actin activate the Mg-ATPase in a hyperbolic manner (KATPase of 1.3 microM). Brain myosin-V also binds with relatively high affinity (compared to other known myosins) to F-actin in the presence of ATP, as assayed by cosedimentation. Digestion of brain myosin-V with calpain yielded a 65-kDa head domain fragment that cosediments with actin in an ATP-sensitive manner and a 80-kDa tail fragment that does not interact with F-actin. The 80-kDa fragment results from cleavage one residue beyond the proline-, glutamate-, serine-, threonine-rich region. Our data indicate that the Mg-ATPase cycle of brain myosin-V is tightly regulated by Ca2+, probably via direct binding to the calmodulin light chains in the neck domain, which like brush border myosin-I, results in partial (approximately 30%) dissociation of the calmodulin associated with brain myosin-V. The effect of Ca2+ binding, which appears to relieve suppression by the neck domain, can be mimicked by calpain cleavage near the head/neck junction.

Functional expression of mammalian myosin I beta: analysis of its motor activity.

The motor function of vertebrate unconventional myosins is not well understood. In this study, we initiated the baculovirus expression system to characterize a novel myosin I from bovine adrenal gland that we had previously cloned [Zhu, T., & Ikebe, M. (1994) FEBS Lett. 339, 31-36], which is classified as myosin I beta. The expressed myosin I beta was well extracted when calmodulin was coexpressed in Sf9 cells. The recombinant myosin I beta cosedimented with actin in an ATP dependent manner. The purified myosin I beta was composed of one heavy chain and three calmodulins. The electron microscopic image of myosin I beta confirmed its single-headed structure with a short tail, which is similar to that of brush border myosin I (BBMI). Myosin I beta showed high K+,EDTA--ATPase activity (approximately 0.14 mumol/min/mg) and Ca(2+)-ATPase activity (approximately 0.32 mumol/min/mg), and the KCl/pH dependence of these activities was different from that of conventional myosin. Mg(2+)-ATPase activity of myosin I beta alone was increased above pCa 6, while the actin dependent activity was not affected by Ca2+. Actin sliding velocity of myosin I beta in the absence of Ca2+ was 0.3-0.5 microns/s at 25 degrees C, which is much greater than that of BBMI (< 0.05 microns/s). The actin sliding activity was abolished above pCa 6, and the sliding activity was restored when exogenous calmodulin was added in the absence of Ca2+. Within similar Ca2+ concentrations, one of the three calmodulins was dissociated from myosin I beta. The results suggest that Ca2+ dependent association of calmodulin may function as a regulatory mechanism of myosin I beta motor activity and that the motor activity of mammalian myosin I is largely different among distinct myosin I isoforms.

A 32 degree tail swing in brush border myosin I on ADP release.

Brush border myosin I (BBMI) is a single-headed, unconventional myosin from intestinal microvilli, composed of a heavy chain of relative molecular mass 119,000 (M(r) 119K) and three calmodulin light chains. Although believed to have a largely structural role, it exhibits the normal actin-activated ATPase and motility properties of a member of the myosin superfamily. Here we present three-dimensional maps of BBMI-decorated actin filaments with and without bound MgADP. While the motor domain remains in a state similar to rigor, the light-chain-binding domain swings through approximately 32 degrees, resulting in a approximately 50-A movement at the end of the region visualized (the second calmodulin light chain). This could correspond to approximately 72-A movement of the entire domain. Although qualitatively similar to the movement observed in myosin II, the magnitude of the change is sufficiently different to suggest that structural changes during the actomyosin ATPase cycle differ among myosins, possibly reflecting adaptation for specialized functional demands.

Identification of Brush Border Myosin-I in Liver and Testis

Brush border myosin-I, or BBMI, constitutes the lateral links that connect in intestinal microvilli the core bundle of actin filaments to the membrane. Although related molecules have been identified in other higher eukaryotic tissues, northern blot analysis has indicated that the distribution of this particular myosin-I isoform is restricted essentially to intestine. Using reverse transcriptase polymerase chain reaction we have identified BBMI in a wide range of tissues including liver and testis. Our results also indicate that in testis the BBMI gene might be alternatively spliced.