Lynne Coluccio

Boston University | BU · Department of Physiology and Biophysics

Pancreatic β-cells secrete insulin, which controls blood glucose levels, and defects in insulin secretion are responsible for diabetes mellitus. The actin cytoskeleton and some myosins support insulin granule trafficking and release, although a role for the class I myosin Myo1b, an actin- and membrane-associated load-sensitive motor, in insulin bio...

Myosins constitute a superfamily of actin-based molecular motor proteins that mediates a variety of cellular activities including muscle contraction, cell migration, intracellular transport, the formation of membrane projections, cell adhesion, and cell signaling. The 12 myosin classes that are expressed in humans share sequence similarities especi...

Myosins are molecular motors that use the energy from ATP hydrolysis to move and exert tension on actin filaments. Although the best-known myosin is myosin II, which powers skeletal muscle contraction, there are at least two dozen classes of myosins, and cells generally express multiple isoforms. Myosins are involved in multiple cellular activities...

Myosin X (Myo10), an actin-associated molecular motor, has a clear role in filopodia induction and cell migration in vitro, but its role in vivo in mammals is not well understood. Here, we investigate the role of Myo10 in melanocyte lineage and melanoma induction. We found that Myo10 knockout (Myo10KO) mice exhibit a white spot on their belly cause...

Amino acid transporters (AATers) in the brush border of the apical plasma membrane (APM) of renal proximal tubule (PT) cells mediate amino acid transport (AAT). We found that the membrane-associated class I myosin myosin 1b (Myo1b) localized at the apical brush border membrane of PTs. In opossum kidney (OK) 3B/2 epithelial cells, which are derived...

Cooperation between cadherins and the actin cytoskeleton controls the formation and maintenance of cell-cell adhesions in epithelia. We find that the molecular motor protein myosin-1c (Myo1c) regulates the dynamic stability of E-cadherin-based cell-cell contacts. In Myo1c-depleted MDCK cells, E-cadherin localization was disorganized and lateral mem...

Here, we report that the natural compound pentachloropseudilin (PClP) acts as a reversible and allosteric inhibitor of myosin ATPase and motor activity. IC50 values are in the range from 1 to 5 μm for mammalian class-1 myosins and greater than 90 μm for class-2 and class-5 myosins, and no inhibition was observed with class-6 and class-7 myosins. We...

Three heterozygous missense mutations in the motor domain of myosin 1c (Myo1c), which mediates adaptation in the inner ear, are associated with bilateral sensorineural hearing loss in humans. With transient kinetic analyses, steady-state ATPase and motility assays, and homology modeling, we studied the interaction of these mutants with nucleotide a...

The actin cytoskeleton is regulated by a variety of actin-binding proteins including those constituting the tropomyosin family. Tropomyosins are coiled-coil dimers that bind along the length of actin filaments. In muscles, tropomyosin regulates the interaction of actin-containing thin filaments with myosin-containing thick filaments to allow contra...

Myosin 1b (Myo1b), a class I myosin, is a widely expressed, single-headed, actin-associated molecular motor. Transient kinetic and single-molecule studies indicate that it is kinetically slow and responds to tension. Localization and subcellular fractionation studies indicate that Myo1b associates with the plasma membrane and certain subcellular or...

Myo1c is one of eight members of the mammalian myosin I family of actin-associated molecular motors. In stereocilia of the hair cells in the inner ear, Myo1c presumably serves as the adaptation motor, which regulates the opening and closing of transduction channels. Although there is conservation of sequence and structure among all myosins in the N...

To investigate the interaction of mammalian class I myosin, Myo1c, with its light chain calmodulin, we expressed (with calmodulin) truncation mutants consisting of the Myo1c motor domain followed by 0-4 presumed calmodulin-binding (IQ) domains (Myo1c (0IQ)-Myo1c (4IQ)). The amount of calmodulin associating with the Myo1c heavy chain increased with...

The class I myosin Myo1c is a mediator of adaptation of mechanoelectrical transduction in the stereocilia of the inner ear. Adaptation, which is strongly affected by Ca²⁺, permits hair cells under prolonged stimuli to remain sensitive to new stimuli. Using a Myo1c fragment (motor domain and one IQ domain with associated calmodulin), with biochemica...

Few would have predicted 20 years ago that myosins constitute a superfamily with at least two-dozen classes and that these molecular motors are involved in a multitude of intracellular activities including cell division, cell movement, intracellular transport and signal transduction. Application of state-of-the-art cellular and molecular biological...

Our understanding of the structure-function relationships in the myosin super family is largely based on studies of conventional myosin II isolated from striated muscle.The background to the elucidation of the mechanism of the actin-activated myosin II ATPase is described in detail and variations in kinetic properties within the myosin II class are...

Myosins are motor proteins that use the energy derived from ATP hydrolysis to move unidirectionally along actin filament tracks within the cell. Myosin VI appears to be unique, because unlike all the other myosins so far characterised, it moves backwards towards the minus end of actin filaments. Within cells myosin VI is found in distinct locations...

In mammals, three different isoforms of nonmuscle myosin II, II-A,II-B and II-C, are widely distributed throughout the entire organism. while a few cells contain a single isoform, most contain more than one,including isoforms generated by alternative splicing. In humans, these isoforms are encoded by three different genes, MYH9 (II-A), MYH10 and MY...

The major motor protein in all hollow organs, except the heart, is smooth-muscle myosin II (SmM) and the emphasis of this chapter is on the function of SmM in differentiated smooth muscle. A sliding-filament mechanism is assumed for smooth muscle, as in striated muscle, but there are differences in smooth-muscle thick filaments with respect to asse...

Class I myosins are single-headed molecular mechano-enzymes, or motors, that translocate actin filaments in vitro. They constitute the largest group of unconventional myosins. Found in many different organisms from protists and yeast to vertebrates, they are often associated with membranes. Class I myosins are diverse in structure, regulation and f...

MyosinX(Myo10)is aMyTH4-FERMmyosinspecific to chordatesandrelated organisms. It is unique in that its tail contains three Pleckstrin Homology (PH) domains. Myo10 is expressed in most cells and tissues, although at relatively low levels. Myo10 localizes to regions of dynamic actin such as the tips of filopodia, where it may function as part of a fil...

In this chapter, a broad overview of the myosin family of actin-based motors with respect to diversity of myosin-domain structures and cellular functions will be outlined.It is meant as a backdrop for the more detailed discussions of the various myosin classes found in the chapters that follow. This is not intended to be a comprehensive review;rath...

The class III myosins are distinguished from other myosin classes by the kinase at their N- terminus, but resemble other myosins in that ahey possess a typical conserved motor domain, a neck domain with IQ motifs and variable tails. Myosin III is expressed predominantly in sensory cells in both invertebrates and vertebrates where it localizes to el...

Mutations of MYO15A are associated with deafness in humans. In the shaker 2 mouse (Myo15a sh2), a missense mutation in the motor domain of myosin XVa causes deafness and circling behavior, and is morphologically characterized by abnormally short stereocilia bundles on the apical surface of inner ear hair cells that also lack the characteristic stai...

There are three classes of myosins in plants: myosins VIII, XI, and XIII. Myosins VIII and XI are widely distributed and found not only in higher plants, but also in Chlamydomonas, while myosin XIII is found only in Acetabularia. Biochemical studies have been done mainly on myosin XI, which is the most abundantly expressed myosin in plants.

Structural studies of the class I myosin, MyoE, led to the predictions that loop 4, a surface loop near the actin-binding region that is longer in class I myosins than in other myosin subclasses, might limit binding of myosins I to actin when actin-binding proteins, like tropomyosin, are present, and might account for the exclusion of myosin I from...

Loop 1, a flexible surface loop in the myosin motor domain, comprises in part the transducer region that lies near the nucleotide-binding site and is proposed from structural studies to be responsible for the kinetic tuning of product release following ATP hydrolysis (1). Biochemical studies have shown that loop 1 affects the affinity of actin-myos...

The class I myosin, Myo1b, is a calmodulin- and actin-associated molecular motor widely expressed in mammalian tissues. Analytical ultracentrifugation studies indicate that Myo1b purified from rat liver has a Stokes radius of 6.7 nm and a sedimentation coefficient, s(20,w), of 7.0 S with a predicted molar mass of 213 kg/mol. These results indicate...

We have used an optical tweezers-based apparatus to perform single molecule mechanical experiments using the unconventional myosins, Myo1b and Myo1c. The single-headed nature and slow ATPase kinetics of these myosins make them ideal for detailed studies of the molecular mechanism of force generation by acto-myosin. Myo1c exhibits several features t...

The molecular motor, Myo1c, a member of the myosin family, is widely expressed in vertebrate tissues. Its presence at strategic places in the stereocilia of the hair cells in the inner ear and studies using transgenic mice expressing a mutant Myo1c that can be selectively inhibited implicate it as the mediator of slow adaptation of mechanoelectrica...

The movement produced by a small number of myosin molecular motors was measured with nanometre precision using single-molecule fluorescence localisation methods. The positional precision of the measurements was sufficient to reveal fluctuations in sliding velocity due to stochastic interactions between individual myosin motors and the actin filamen...

MI(1IQ) is a complex of calmodulin and an epitope-tagged 85-kDa fragment representing the amino-terminal catalytic motor domain and the first of 6 calmodulin-binding IQ domains of the mammalian myosin I gene, rat myr-1 (130-kDa myosin I or MI(130)). We have determined the transient kinetic parameters that dictate the ATP hydrolysis cycle of mammali...

MYR-1, a mammalian class I myosin, consisting of a heavy chain and 4–6 associated calmodulins, is represented by the 130-kDa myosin I (or MI130) from rat liver. MI130 translocates actin filaments in vitro in a Ca2+-regulated manner. A decrease in motility observed at higher Ca2+ concentrations has been attributed to calmodulin dissociation. To inve...

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...

The biochemical and mechanochemical properties and localization of myosin I suggest the involvement of these small members of the myosin superfamily in some aspects of intracellular motility in higher cells. We have determined by quantitative immunoblotting with isoform-specific antibodies that the 130-kDa myosin I (myr 1 gene product) and 110-kDa...

The 130-kDa myosin I (MI130), product of the myr-1 gene, is one member of the mammalian class I myosins, a group of small, calmodulin-binding mechanochemical molecules of the myosin superfamily that translocate actin filaments. Roles for MI130 are unknown. Our hypothesis is that, as with all myosins, MI130 is designed for a particular function and...

Many types of cellular motility, including muscle contraction, are driven by the cyclical interaction of the motor protein myosin with actin filaments, coupled to the breakdown of ATP. It is thought that myosin binds to actin and then produces force and movement as it 'tilts' or 'rocks' into one or more subsequent, stable conformations. Here we use...

The class I myosins are single-headed, actin-binding, mechanochemical "motor" proteins with heavy chains in the molecular mass range of 110-130 kDa; they do not form filaments. Each myosin I heavy chain is associated with one to six light chains that bind to specific motifs known as IQ domains. In vertebrate myosin I isoforms, the light chain is ca...

Three isoforms of the cytoskeletal-associated, mechanochemical enzymes known as myosin-I have been purified from rat liver; each coisolates with calmodulin. Incubation of the purified myosin-I's with protein kinase C gamma and 32P-ATP results in phosphorylation of the myosin-I heavy chains. After phosphorylation, the myosin-I isoforms bind less rad...

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 essen...

We have previously purified and characterized two myosin-1 isoforms from rat liver (molecular masses 130 kDa and 110 kDa; L. M. Coluccio and C. Conaty (1993) Cell Motil. Cytoskel. 24, 189-199). Here, we describe the purification and characterization from liver of a third myosin-1 (molecular mass 105 kDa) and determine the number of calmodulin molec...

We have recently purified and characterized from rat liver, polypeptides of 110-kDa and 130-kDa which possess several characteristics of myosin-1 [Coluccio and Conaty: Cell Motil. Cytoskeleton 24:189-199, 1993]. What roles these myosin-1 molecules play in hepatocytes is not yet defined. One hypothesis is that they are involved in either intracellul...

Myosin-I refers to a class of proteins with a molecular weight of approximately 110-kDa, which have characteristics of conventional myosin but are unable to form filaments. Previous studies have implicated myosin-I in motile cellular processes including cell migration and phagocytosis. Although the first example of myosin-I in higher eukaryotes was...

The epithelial layer lining the proximal convoluted tubule of mammalian kidney contains a brush border of numerous microvilli. These microvilli appear in structure to be very similar to the microvilli on epithelial cells of the small intestine. Microvilli found in both the small intestine and the proximal convoluted tubules in kidney have a core bu...

In intestinal microvilli, the 110K-calmodulin complex is the major component of the cross-bridges which connect the core bundle of actin filaments to the membrane. Our previous work showed that the 110-kDa polypeptide can be divided into three functional domains: a 78-kDa fragment that contains the ATPase activity and the ATP-reversible F-actin-bin...

Intestinal epithelia have a brush border membrane of numerous microvilli each comprised of a cross-linked core bundle of 15-20 actin filaments attached to the surrounding membrane by lateral cross-bridges; the cross-bridges are tilted with respect to the core bundle. Isolated microvillar cores contain actin (42 kD) and three other major proteins: f...

We present the three-dimensional structure of an actin filament bundle from the sperm of Limulus. The bundle is a motile structure which by changing its twist, converts from a coiled to an extended form. The bundle is composed of actin plus two auxiliary proteins of molecular masses 50 and 60 kD. Fraying the bundle with potassium thiocyanate create...

The 110K-calmodulin complex isolated from intestinal microvilli is an ATPase consisting of one polypeptide chain of 110 kD in association with three to four calmodulin molecules. This complex is presumably the link between the actin filaments in the microvillar core and the surrounding cell membrane. To study its structural regions, we have partial...

Highly purified microvillar 110 kDa polypeptide-calmodulin (110K-cam) complex was confirmed to have ATPase activities characteristic of a myosin. The effect of F-actin on these activities was investigated. The Mg2+-ATPase is activated about 2-fold by F-actin in a dose-dependent fashion, whereas the K+-EDTA-ATPase is inhibited by greater than 90% by...

The 110K-calmodulin complex of intestinal microvilli is believed to be the link between the actin filaments comprising the core bundle and the surrounding cell membrane. Although not the first study describing a purification scheme for the 110K-calmodulin complex, a procedure for the isolation of stable 110K-calmodulin complex both pure and in high...

A 45 kDa actin-binding protein (SU45) has been isolated previously from egg extracts of the Hawaiian sea urchin Tripneustes gratilla by DEAE Sephacel, Sephadex G-75 and hydroxyapatite chromatography. Using pyrene-labelled rabbit skeletal muscle actin, we have found that when SU45 is added to actin in the presence of calcium and the salt concentrati...

Platelet gelsolin (G), a 90,000-mol-wt protein, binds tightly to actin (A) and calcium at low ionic strength to form a 1:2:2 complex, GA2Ca2 (Bryan, J., and M. Kurth, 1984, J. Biol. Chem. 259:7480-7487). Chromatography of actin and gelsolin mixtures in EGTA-containing solutions isolates a stable binary complex, GA1Ca1 (Kurth, M., and J. Bryan, 1984...

Incubation of the isolated acrosomal bundles of Limulus sperm with skeletal muscle actin results in assembly of actin onto both ends of the bundles. These cross-linked bundles of actin filaments taper, thus allowing one to distinguish directly the preferred end for actin assembly from the nonpreferred end; the preferred end is thinner. Incubation w...

Incubation of the isolated acrosomal bundles of Limulus sperm with skeletal muscle actin results in assembly of actin onto both ends of the bundles. Because of the taper of these cross-linked bundles of actin filaments, one can distinguish directly the preferred end for assembly from the nonpreferred end. Loss of growth with time from the nonprefer...

Thyone sperm were demembranated with Triton X-100 and, after washing, extracted with 30 mM Tris at pH 8.0 and 1 mM MgCl2. After the insoluble contaminants were removed by centrifugation, the sperm extract was warmed to 22 degrees C. Actin filaments rapidly assembled and aggregated into bundles when KCl was added to the extract. When we added prefor...

Earlier studies employed colchicine to demonstrate the need for microtubules in the ADH-induced initiation of increased water permeability in toad bladder. We have used colchicine and hydrostatic pressure together to determine whether formed or growing microtubules are required for initiation of the ADH response in Bufo marinus. When ADH and 8,000...

The aggregation properties of column-purified rabbit skeletal myosin at pH 7.0 were investigated as functions of ionic strength, protein concentration, and time. Filaments prepared by dialysis exhibited the same average length and population distribution at 0.10 and 0.15 M KCl at protein concentrations greater than 0.10 mg/ml; similar results were...

Nocodazole is a synthetic antitumor drug that binds rapidly to tubulin. When this drug is applied to toad bladder prior to vasopressin stimulation it inhibits the vasopressin response. A maximum inhibition (68%) is reached with a dose level of 10 micrograms/ml applied one-half hour prior to vasopressin stimulation (20 mU/ml). This compares with an...

Class VII myosins are among the most widely expressed myosins in the animal kingdom. They also have a broad tissue expression. Vertebrates and some invertebrates possess two different myosins VII, myosin VIIa and myosin VIIb, which may differ in their kinetic properties. Defects in myosin VIIa cause phenotypic anomalies in Drosophila, zebrafish, mo...

Myosin V is a two-headed cargo-transporting myosin that is found in most animal genomes. Next to the conventional class II myosins, it is probably the most studied myosin class and is, perhaps, the best characterized in terms of its molecular basis for action. This is directly related to its molecular properties. As a cargo-carrying protein, it has...

Class IX myosins are found in animals from invertebrates to vertebrates. Invertebrates contain a single myosin IX gene, whereas vertebrates contain two myosin IX genes, MYO9A and MYO9B. Mammalian Myo9b, the only class IX myosin studied so far, has unique motor properties. It is the first myosin for which ATP hydrolysis is the rate-limiting step in...

Myosin II, the myosin which has provided the most biochemical and structural data, is dimeric consisting of a long coiled-coil region with the motor domain flexibly attached to the N-terminal end of the coiled-coil. The motor domain (subfragment 1, S1, or cross-bridge) is obtained by proteolytic cleavage of myosin. S1 is a minimal model for the tra...

Myosins are actin-based molecular motors that convert chemical energy released by ATP hydrolysis into directed movement along tracks of actin filaments. They are found in eukaryotes and are implicated in a number of important cell functions, such as nuclear and cell division, transport of molecules, vesicles, and organelles, signal transduction, an...

Sarcomeric myosins are class II myosins, which are expressed in striated muscles and incorporated into the sarcomeric architecture. As such, sarcomeric myosins are the motors of skeletal and cardiac muscle contraction, thus responsible for animal posture and movement as well as blood circulation. Two distinctive features characterize sarcomeric myo...