RESUMO
Molecular motors employ chemical energy to generate unidirectional mechanical output against a track while navigating a chaotic cellular environment, potential disorder on the track, and against Brownian motion. Nevertheless, decades of nanometer-precise optical studies suggest that myosin-5a, one of the prototypical molecular motors, takes uniform steps spanning 13 subunits (36 nm) along its F-actin track. Here, we use high-resolution interferometric scattering microscopy to reveal that myosin takes strides spanning 22 to 34 actin subunits, despite walking straight along the helical actin filament. We show that cumulative angular disorder in F-actin accounts for the observed proportion of each stride length, akin to crossing a river on variably spaced stepping stones. Electron microscopy revealed the structure of the stepping molecule. Our results indicate that both motor and track are soft materials that can adapt to function in complex cellular conditions.
Assuntos
Actinas , Miosina Tipo V , Actinas/química , Miosinas/química , Citoesqueleto de Actina/química , Movimento (Física) , Miosina Tipo V/químicaRESUMO
Myosin-7a is an actin-based motor protein essential for vision and hearing. Mutations of myosin-7a cause type 1 Usher syndrome, the most common and severe form of deafblindness in humans. The molecular mechanisms that govern its mechanochemistry remain poorly understood, primarily because of the difficulty of purifying stable intact protein. Here, we recombinantly produce the complete human myosin-7a holoenzyme in insect cells and characterize its biochemical and motile properties. Unlike the Drosophila ortholog that primarily associates with calmodulin (CaM), we found that human myosin-7a utilizes a unique combination of light chains including regulatory light chain, CaM, and CaM-like protein 4. Our results further reveal that CaM-like protein 4 does not function as a Ca2+ sensor but plays a crucial role in maintaining the lever arm's structural-functional integrity. Using our recombinant protein system, we purified two myosin-7a splicing isoforms that have been shown to be differentially expressed along the cochlear tonotopic axis. We show that they possess distinct mechanoenzymatic properties despite differing by only 11 amino acids at their N termini. Using single-molecule in vitro motility assays, we demonstrate that human myosin-7a exists as an autoinhibited monomer and can move processively along actin when artificially dimerized or bound to cargo adaptor proteins. These results suggest that myosin-7a can serve multiple roles in sensory systems such as acting as a transporter or an anchor/force sensor. Furthermore, our research highlights that human myosin-7a has evolved unique regulatory elements that enable precise tuning of its mechanical properties suitable for mammalian auditory functions.
Assuntos
Actinas , Surdocegueira , Miosina VIIa , Humanos , Actinas/metabolismo , Ligação Proteica , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Miosina VIIa/genética , Miosina VIIa/metabolismo , Calmodulina/metabolismo , Proteínas de Ligação ao Cálcio/metabolismoRESUMO
Recent genomic studies reported that 90 to 95% of human genes can undergo alternative splicing, by which multiple isoforms of proteins are synthesized. However, the functional consequences of most of the isoforms are largely unknown. Here, we report a novel alternatively spliced isoform of nonmuscle myosin IIA (NM IIA), called NM IIA2, which is generated by the inclusion of 21 amino acids near the actin-binding region (loop 2) of the head domain of heavy chains. Expression of NM IIA2 is found exclusively in the brain tissue, where it reaches a maximum level at 24 h during the circadian rhythm. The actin-dependent Mg2+-ATPase activity and in vitro motility assays reveal that NM IIA2 lacks its motor activities but localizes with actin filaments in cells. Interestingly, NM IIA2 can also make heterofilaments with NM IIA0 (noninserted isoform of NM IIA) and can retard the in vitro motility of NM IIA, when the two are mixed. Altogether, our findings provide the functional importance of a previously unknown alternatively spliced isoform, NM IIA2, and its potential physiological role in regulating NM IIA activity in the brain.
Assuntos
Processamento Alternativo , Encéfalo , Miosina não Muscular Tipo IIA , Humanos , Actinas/metabolismo , Encéfalo/metabolismo , Cadeias Pesadas de Miosina/química , Cadeias Pesadas de Miosina/metabolismo , Miosina não Muscular Tipo IIA/química , Miosina não Muscular Tipo IIA/genética , Miosina não Muscular Tipo IIA/metabolismo , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Ritmo Circadiano , ATPase de Ca(2+) e Mg(2+)/metabolismo , Especificidade de ÓrgãosRESUMO
Aging is associated with cognitive decline and is the main risk factor for a myriad of conditions including neurodegeneration and stroke. Concomitant with aging is the progressive accumulation of misfolded proteins and loss of proteostasis. Accumulation of misfolded proteins in the endoplasmic reticulum (ER) leads to ER stress and activation of the unfolded protein response (UPR). The UPR is mediated, in part, by the eukaryotic initiation factor 2α (eIF2α) kinase protein kinase R-like ER kinase (PERK). Phosphorylation of eIF2α reduces protein translation as an adaptive mechanism but this also opposes synaptic plasticity. PERK, and other eIF2α kinases, have been widely studied in neurons where they modulate both cognitive function and response to injury. The impact of astrocytic PERK signaling in cognitive processes was previously unknown. To examine this, we deleted PERK from astrocytes (AstroPERKKO ) and examined the impact on cognitive functions in middle-aged and old mice of both sexes. Additionally, we tested the outcome following experimental stroke using the transient middle cerebral artery occlusion (MCAO) model. Tests of short-term and long-term learning and memory as well as of cognitive flexibility in middle-aged and old mice revealed that astrocytic PERK does not regulate these processes. Following MCAO, AstroPERKKO had increased morbidity and mortality. Collectively, our data demonstrate that astrocytic PERK has limited impact on cognitive function and has a more prominent role in the response to neural injury.
Assuntos
Astrócitos , Aprendizagem , Acidente Vascular Cerebral , eIF-2 Quinase , Animais , Feminino , Masculino , Camundongos , Retículo Endoplasmático , Proteínas Quinases , eIF-2 Quinase/metabolismoRESUMO
Megakaryocytes (MKs), the precursor cells for platelets, migrate from the endosteal niche of the bone marrow (BM) toward the vasculature, extending proplatelets into sinusoids, where circulating blood progressively fragments them into platelets. Nonmuscle myosin IIA (NMIIA) heavy chain gene (MYH9) mutations cause macrothrombocytopenia characterized by fewer platelets with larger sizes leading to clotting disorders termed myosin-9-related disorders (MYH9-RDs). MYH9-RD patient MKs have proplatelets with thicker and fewer branches that produce fewer and larger proplatelets, which is phenocopied in mouse Myh9-RD models. Defective proplatelet formation is considered to be the principal mechanism underlying the macrothrombocytopenia phenotype. However, MYH9-RD patient MKs may have other defects, as NMII interactions with actin filaments regulate physiological processes such as chemotaxis, cell migration, and adhesion. How MYH9-RD mutations affect MK migration and adhesion in BM or NMIIA activity and assembly prior to proplatelet production remain unanswered. NMIIA is the only NMII isoform expressed in mature MKs, permitting exploration of these questions without complicating effects of other NMII isoforms. Using mouse models of MYH9-RD (NMIIAR702C+/-GFP+/-, NMIIAD1424N+/-, and NMIIAE1841K+/-) and in vitro assays, we investigated MK distribution in BM, chemotaxis toward stromal-derived factor 1, NMIIA activity, and bipolar filament assembly. Results indicate that different MYH9-RD mutations suppressed MK migration in the BM without compromising bipolar filament formation but led to divergent adhesion phenotypes and NMIIA contractile activities depending on the mutation. We conclude that MYH9-RD mutations impair MK chemotaxis by multiple mechanisms to disrupt migration toward the vasculature, impairing proplatelet release and causing macrothrombocytopenia.
Assuntos
Movimento Celular , Perda Auditiva Neurossensorial/complicações , Megacariócitos/patologia , Mutação , Cadeias Pesadas de Miosina/genética , Miosina não Muscular Tipo IIA/genética , Trombocitopenia/congênito , Trombocitopenia/patologia , Animais , Quimiocina CXCL12/genética , Quimiocina CXCL12/metabolismo , Megacariócitos/metabolismo , Camundongos , Camundongos Knockout , Trombocitopenia/complicações , Trombocitopenia/etiologia , Trombocitopenia/metabolismoRESUMO
The Arf GTPase-activating protein (Arf GAP) with SH3 domain, ankyrin repeat and PH domain 1 (ASAP1) establishes a connection between the cell membrane and the cortical actin cytoskeleton. The formation, maintenance, and turnover of actin filaments and bundles in the actin cortex are important for cell adhesion, invasion, and migration. Here, using actin cosedimentation, polymerization, and depolymerization assays, along with total internal reflection fluorescence (TIRF), confocal, and EM analyses, we show that the N-terminal N-BAR domain of ASAP1 directly binds to F-actin. We found that ASAP1 homodimerization aligns F-actin in predominantly unipolar bundles and stabilizes them against depolymerization. Furthermore, the ASAP1 N-BAR domain moderately reduced the spontaneous polymerization of G-actin. The overexpression of the ASAP1 BAR-PH tandem domain in fibroblasts induced the formation of actin-filled projections more effectively than did full-length ASAP1. An ASAP1 construct that lacked the N-BAR domain failed to induce cellular projections. Our results suggest that ASAP1 regulates the dynamics and the formation of higher-order actin structures, possibly through direct binding to F-actin via its N-BAR domain. We propose that ASAP1 is a hub protein for dynamic protein-protein interactions in mechanosensitive structures, such as focal adhesions, invadopodia, and podosomes, that are directly implicated in oncogenic events. The effect of ASAP1 on actin dynamics puts a spotlight on its function as a central signaling molecule that regulates the dynamics of the actin cytoskeleton by transmitting signals from the plasma membrane.
Assuntos
Citoesqueleto de Actina/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Camundongos , Células NIH 3T3 , Ligação Proteica , Transdução de SinaisRESUMO
Addition of 1 mM ATP substantially reduces the light scattering of solutions of polymerized unphosphorylated nonmuscle myosin IIs (NM2s), and this is reversed by phosphorylation of the regulatory light chain (RLC). It has been proposed that these changes result from substantial depolymerization of unphosphorylated NM2 filaments to monomers upon addition of ATP, and filament repolymerization upon RLC-phosphorylation. We now show that the differences in myosin monomer concentration of RLC-unphosphorylated and -phosphorylated recombinant mammalian NM2A, NM2B, and NM2C polymerized in the presence of ATP are much too small to explain their substantial differences in light scattering. Rather, we find that the decrease in light scattering upon addition of ATP to polymerized unphosphorylated NM2s correlates with the formation of dimers, tetramers, and hexamers, in addition to monomers, an increase in length, and decrease in width of the bare zones of RLC-unphosphorylated filaments. Both effects of ATP addition are reversed by phosphorylation of the RLC. Our data also suggest that, contrary to previous models, assembly of RLC-phosphorylated NM2s at physiological ionic strength proceeds from folded monomers to folded antiparallel dimers, tetramers, and hexamers that unfold and polymerize into antiparallel filaments. This model could explain the dynamic relocalization of NM2 filaments in vivo by dephosphorylation of RLC-phosphorylated filaments, disassembly of the dephosphorylated filaments to folded monomers, dimers, and small oligomers, followed by diffusion of these species, and reassembly of filaments at the new location following rephosphorylation of the RLC.
Assuntos
Trifosfato de Adenosina/química , Modelos Moleculares , Cadeias Pesadas de Miosina/química , Miosina Tipo II/química , Multimerização Proteica , Trifosfato de Adenosina/metabolismo , Animais , Humanos , Camundongos , Cadeias Pesadas de Miosina/metabolismo , Miosina Tipo II/metabolismo , FosforilaçãoRESUMO
Nonmuscle myosin 2 (NM2) has three paralogs in mammals, NM2A, NM2B, and NM2C, which have both unique and overlapping functions in cell migration, formation of cell-cell adhesions, and cell polarity. Their assembly into homo- and heterotypic bipolar filaments in living cells is primarily regulated by phosphorylation of the N-terminally bound regulatory light chain. Here, we present evidence that the equilibrium between these filaments and single NM2A and NM2B molecules can be controlled via S100 calcium-binding protein interactions and phosphorylation at the C-terminal end of the heavy chains. Furthermore, we show that in addition to S100A4, other members of the S100 family can also mediate disassembly of homotypic NM2A filaments. Importantly, these proteins can selectively remove NM2A molecules from heterotypic filaments. We also found that tail phosphorylation (at Ser-1956 and Ser-1975) of NM2B by casein kinase 2, as well as phosphomimetic substitutions at sites targeted by protein kinase C (PKC) and transient receptor potential cation channel subfamily M member 7 (TRPM7), down-regulates filament assembly in an additive fashion. Tail phosphorylation of NM2A had a comparatively minor effect on filament stability. S100 binding and tail phosphorylation therefore preferentially disassemble NM2A and NM2B, respectively. These two distinct mechanisms are likely to contribute to the temporal and spatial sorting of the two NM2 paralogs within heterotypic filaments. The existence of multiple NM2A-depolymerizing S100 paralogs offers the potential for diverse regulatory inputs modulating NM2A filament disassembly in cells and provides functional redundancy under both physiological and pathological conditions.
Assuntos
Miosina não Muscular Tipo IIA/metabolismo , Miosina não Muscular Tipo IIB/metabolismo , Isoformas de Proteínas/metabolismo , Proteínas S100/metabolismo , Animais , Caseína Quinase II/genética , Caseína Quinase II/metabolismo , Citoesqueleto/metabolismo , Proteínas de Fluorescência Verde/genética , Humanos , Mutação , Miosina não Muscular Tipo IIA/química , Miosina não Muscular Tipo IIB/química , Ressonância Magnética Nuclear Biomolecular , Fosforilação , Ligação Proteica , Proteínas Serina-Treonina Quinases/metabolismo , Células Sf9 , Canais de Cátion TRPM/metabolismoRESUMO
Mammalian cells express three Class II nonmuscle myosins (NM): NM2A, NM2B, and NM2C. The three NM2s have well established essential roles in cell motility, adhesion, and cytokinesis and less well defined roles in vesicle transport and other processes that would require association of NM2s with cell membranes. Previous evidence for the mechanism of NM2-membrane association includes direct interaction of NM2s with membrane lipids and indirect interaction by association of NM2s with membrane-bound F-actin or peripheral membrane proteins. Direct binding of NM2s to phosphatidylserine-liposomes, but not to phosphatidylcholine-liposomes, has been reported, but the molecular basis of the interaction between NM2s and acidic phospholipids has not been previously investigated. We now show that filamentous, full-length NM2A, NM2B, and NM2C and monomeric, non-filamentous heavy meromyosin bind to liposomes containing one or more acidic phospholipids (phosphatidylserine, phosphatidylinositol 4,5-diphosphate, and phosphatidylinositol 3,4,5-triphosphate) but do not bind to 100% phosphatidylcholine-liposomes. Binding of NM2s to acidic liposomes occurs predominantly through interaction of the liposomes with the regulatory light chain (RLC) binding site in the myosin heavy chain with concomitant dissociation of the RLC. Phosphorylation of myosin-bound RLC by myosin light chain kinase substantially inhibits binding to liposomes of both filamentous NM2 and non-filamentous heavy meromyosin; the addition of excess unbound RLC, but not excess unbound essential light chain, competes with liposome binding. Consistent with the in vitro data, we show that endogenous and expressed NM2A associates with the plasma membrane of HeLa cells and fibrosarcoma cells independently of F-actin.
Assuntos
Membrana Celular/metabolismo , Cadeias Leves de Miosina/metabolismo , Miosina Tipo II/metabolismo , Fosfolipídeos/metabolismo , Actinas/química , Actinas/genética , Actinas/metabolismo , Membrana Celular/química , Membrana Celular/genética , Células HeLa , Humanos , Lipossomos/química , Cadeias Leves de Miosina/química , Cadeias Leves de Miosina/genética , Miosina Tipo II/química , Miosina Tipo II/genética , Fosfolipídeos/químicaRESUMO
Inherited deafness is clinically and genetically heterogeneous. We recently mapped DFNB86, a locus associated with nonsyndromic deafness, to chromosome 16p. In this study, whole-exome sequencing was performed with genomic DNA from affected individuals from three large consanguineous families in which markers linked to DFNB86 segregate with profound deafness. Analyses of these data revealed homozygous mutation c.208G>T (p.Asp70Tyr) or c.878G>C (p.Arg293Pro) in TBC1D24 as the underlying cause of deafness in the three families. Sanger sequence analysis of TBC1D24 in an additional large family in which deafness segregates with DFNB86 identified the c.208G>T (p.Asp70Tyr) substitution. These mutations affect TBC1D24 amino acid residues that are conserved in orthologs ranging from fruit fly to human. Neither variant was observed in databases of single-nucleotide variants or in 634 chromosomes from ethnically matched control subjects. TBC1D24 in the mouse inner ear was immunolocalized predominantly to spiral ganglion neurons, indicating that DFNB86 deafness might be an auditory neuropathy spectrum disorder. Previously, six recessive mutations in TBC1D24 were reported to cause seizures (hearing loss was not reported) ranging in severity from epilepsy with otherwise normal development to epileptic encephalopathy resulting in childhood death. Two of our four families in which deafness segregates with mutant alleles of TBC1D24 were available for neurological examination. Cosegregation of epilepsy and deafness was not observed in these two families. Although the causal relationship between genotype and phenotype is not presently understood, our findings, combined with published data, indicate that recessive alleles of TBC1D24 can cause either epilepsy or nonsyndromic deafness.
Assuntos
Proteínas de Transporte/genética , Epilepsia/genética , Mutação , Alelos , Sequência de Aminoácidos , Cromossomos Humanos Par 16/genética , Consanguinidade , Surdez/genética , Exoma , Éxons , Feminino , Proteínas Ativadoras de GTPase , Genes Recessivos , Loci Gênicos , Estudo de Associação Genômica Ampla , Heterozigoto , Homozigoto , Humanos , Masculino , Proteínas de Membrana , Dados de Sequência Molecular , Proteínas do Tecido Nervoso , Paquistão , Linhagem , Fenótipo , Polimorfismo de Nucleotídeo Único , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Análise de Sequência de DNARESUMO
Myosin-10 is an actin-based molecular motor that participates in essential intracellular processes such as filopodia formation/extension, phagocytosis, cell migration, and mitotic spindle maintenance. To study this motor protein's mechano-chemical properties, we used a recombinant, truncated form of myosin-10 consisting of the first 936 amino acids, followed by a GCN4 leucine zipper motif, to force dimerization. Negative-stain electron microscopy reveals that the majority of molecules are dimeric with a head-to-head contour distance of â¼50 nm. In vitro motility assays show that myosin-10 moves actin filaments smoothly with a velocity of â¼310 nm/s. Steady-state and transient kinetic analysis of the ATPase cycle shows that the ADP release rate (â¼13 s(-1)) is similar to the maximum ATPase activity (â¼12-14 s(-1)) and therefore contributes to rate limitation of the enzymatic cycle. Single molecule optical tweezers experiments show that under intermediate load (â¼0.5 pN), myosin-10 interacts intermittently with actin and produces a power stroke of â¼17 nm, composed of an initial 15-nm and subsequent 2-nm movement. At low optical trap loads, we observed staircase-like processive movements of myosin-10 interacting with the actin filament, consisting of up to six â¼35-nm steps per binding interaction. We discuss the implications of this load-dependent processivity of myosin-10 as a filopodial transport motor.
Assuntos
Actinas/fisiologia , Cadeias Pesadas de Miosina/fisiologia , Actinas/química , Difosfato de Adenosina/metabolismo , Sequência de Aminoácidos , Animais , Fenômenos Biomecânicos , Bovinos , Técnicas In Vitro , Cinética , Microscopia Eletrônica , Microscopia de Fluorescência , Modelos Biológicos , Modelos Moleculares , Dados de Sequência Molecular , Cadeias Pesadas de Miosina/química , Cadeias Pesadas de Miosina/genética , Subfragmentos de Miosina/química , Subfragmentos de Miosina/genética , Subfragmentos de Miosina/fisiologia , Pinças Ópticas , Domínios e Motivos de Interação entre Proteínas , Estrutura Quaternária de Proteína , Pseudópodes/fisiologia , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismoRESUMO
Unconventional myosin 15 is a molecular motor expressed in inner ear hair cells that transports protein cargos within developing mechanosensory stereocilia. Mutations of myosin 15 cause profound hearing loss in humans and mice; however, the properties of this motor and its regulation within the stereocilia organelle are unknown. To address these questions, we expressed a subfragment 1-like (S1) truncation of mouse myosin 15, comprising the predicted motor domain plus three light-chain binding sites. Following unsuccessful attempts to express functional myosin 15-S1 using the Spodoptera frugiperda (Sf9)-baculovirus system, we discovered that coexpression of the muscle-myosin-specific chaperone UNC45B, in addition to the chaperone heat-shock protein 90 (HSP90) significantly increased the yield of functional protein. Surprisingly, myosin 15-S1 did not bind calmodulin with high affinity. Instead, the IQ domains bound essential and regulatory light chains that are normally associated with class II myosins. We show that myosin 15-S1 is a barbed-end-directed motor that moves actin filaments in a gliding assay (â¼ 430 nm · s(-1) at 30 °C), using a power stroke of 7.9 nm. The maximum ATPase rate (k(cat) â¼ 6 s(-1)) was similar to the actin-detachment rate (k(det) = 6.2 s(-1)) determined in single molecule optical trapping experiments, indicating that myosin 15-S1 was rate limited by transit through strongly actin-bound states, similar to other processive myosin motors. Our data further indicate that in addition to folding muscle myosin, UNC45B facilitates maturation of an unconventional myosin. We speculate that chaperone coexpression may be a simple method to optimize the purification of other myosin motors from Sf9 insect cells.
Assuntos
Miosinas/isolamento & purificação , Miosinas/metabolismo , Estereocílios/metabolismo , Actinas/metabolismo , Sequência de Aminoácidos , Animais , Calmodulina/metabolismo , Proteínas de Choque Térmico HSP90/genética , Proteínas de Choque Térmico HSP90/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Camundongos , Chaperonas Moleculares , Dados de Sequência Molecular , Cadeias Leves de Miosina/metabolismo , Subfragmentos de Miosina/genética , Subfragmentos de Miosina/isolamento & purificação , Subfragmentos de Miosina/metabolismo , Miosinas/genética , Pinças Ópticas , Dobramento de Proteína , Transporte Proteico , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/isolamento & purificação , Proteínas Recombinantes de Fusão/metabolismo , Homologia de Sequência de Aminoácidos , Células Sf9 , SpodopteraRESUMO
Perrault syndrome is a genetically and clinically heterogeneous autosomal-recessive condition characterized by sensorineural hearing loss and ovarian failure. By a combination of linkage analysis, homozygosity mapping, and exome sequencing in three families, we identified mutations in CLPP as the likely cause of this phenotype. In each family, affected individuals were homozygous for a different pathogenic CLPP allele: c.433A>C (p.Thr145Pro), c.440G>C (p.Cys147Ser), or an experimentally demonstrated splice-donor-site mutation, c.270+4A>G. CLPP, a component of a mitochondrial ATP-dependent proteolytic complex, is a highly conserved endopeptidase encoded by CLPP and forms an element of the evolutionarily ancient mitochondrial unfolded-protein response (UPR(mt)) stress signaling pathway. Crystal-structure modeling suggests that both substitutions would alter the structure of the CLPP barrel chamber that captures unfolded proteins and exposes them to proteolysis. Together with the previous identification of mutations in HARS2, encoding mitochondrial histidyl-tRNA synthetase, mutations in CLPP expose dysfunction of mitochondrial protein homeostasis as a cause of Perrault syndrome.
Assuntos
Proteases Dependentes de ATP/genética , Endopeptidase Clp/genética , Exoma/genética , Genes Recessivos , Disgenesia Gonadal 46 XX/etiologia , Perda Auditiva Neurossensorial/etiologia , Mitocôndrias/enzimologia , Mutação/genética , Proteases Dependentes de ATP/metabolismo , Trifosfato de Adenosina/metabolismo , Adolescente , Adulto , Feminino , Homozigoto , Humanos , Hibridização In Situ , Masculino , Mitocôndrias/genética , Linhagem , Fenótipo , Adulto JovemRESUMO
Nonmuscle myosin IIs (NM IIs) are a group of molecular motors involved in a wide variety of cellular processes including cytokinesis, migration, and control of cell morphology. There are three paralogs of the NM II heavy chain in humans (IIA, IIB, and IIC), each encoded by a separate gene. These paralogs are expressed at different levels according to cell type and have different roles and intracellular distributions in vivo. Most previous studies on NM II used tissue-purified protein or expressed fragments of the molecule, which presents potential drawbacks for characterizing individual paralogs of the intact protein in vitro. To circumvent current limitations and approach their native properties, we have successfully expressed and purified the three full-length human NM II proteins with their light chains, using the baculovirus/Sf9 system. The enzymatic and structural properties of the three paralogs were characterized. Although each NM II is capable of forming bipolar filaments, those formed by IIC tend to contain fewer constituent molecules than those of IIA and IIB. All paralogs adopt the compact conformation in the presence of ATP. Phosphorylation of the regulatory light chain leads to assembly into filaments, which bind to actin in the presence of ATP. The nature of interactions with actin filaments is shown with different paralogs exhibiting different actin binding behaviors under equivalent conditions. The data show that although NM IIA and IIB form filaments with similar properties, NM IIC forms filaments that are less well suited to roles such as tension maintenance within the cell.
Assuntos
Proteínas Motores Moleculares/química , Proteínas Motores Moleculares/metabolismo , Cadeias Pesadas de Miosina/química , Cadeias Pesadas de Miosina/metabolismo , Miosina Tipo II/química , Miosina Tipo II/metabolismo , Miosina não Muscular Tipo IIB/química , Miosina não Muscular Tipo IIB/metabolismo , Trifosfato de Adenosina/química , Trifosfato de Adenosina/genética , Trifosfato de Adenosina/metabolismo , Animais , Humanos , Proteínas Motores Moleculares/genética , Cadeias Pesadas de Miosina/genética , Miosina Tipo II/genética , Miosina não Muscular Tipo IIB/genética , Fosforilação/fisiologia , Coelhos , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Células Sf9 , SpodopteraRESUMO
Nonmuscle myosin IIB (NMIIB) is a cytoplasmic myosin, which plays an important role in cell motility by maintaining cortical tension. It forms bipolar thick filaments with ~14 myosin molecule dimers on each side of the bare zone. Our previous studies showed that the NMIIB is a moderately high duty ratio (~20-25%) motor. The ADP release step (~0.35 s(-1)) of NMIIB is only ~3 times faster than the rate-limiting phosphate release (0.13 ± 0.01 s(-1)). The aim of this study was to relate the known in vitro kinetic parameters to the results of single molecule experiments and to compare the kinetic and mechanical properties of single- and double-headed myosin fragments and nonmuscle IIB thick filaments. Examination of the kinetics of NMIIB interaction with actin at the single molecule level was accomplished using total internal reflection fluorescence (TIRF) with fluorescence imaging with 1-nm accuracy (FIONA) and dual-beam optical trapping. At a physiological ATP concentration (1 mm), the rate of detachment of the single-headed and double-headed molecules was similar (~0.4 s(-1)). Using optical tweezers we found that the power stroke sizes of single- and double-headed heavy meromyosin (HMM) were each ~6 nm. No signs of processive stepping at the single molecule level were observed in the case of NMIIB-HMM in optical tweezers or TIRF/in vitro motility experiments. In contrast, robust motility of individual fluorescently labeled thick filaments of full-length NMIIB was observed on actin filaments. Our results are in good agreement with the previous steady-state and transient kinetic studies and show that the individual nonprocessive nonmuscle myosin IIB molecules form a highly processive unit when polymerized into filaments.
Assuntos
Subfragmentos de Miosina/química , Miosina não Muscular Tipo IIB/metabolismo , Actinas/química , Animais , Biofísica/métodos , Bovinos , Movimento Celular , Galinhas , Humanos , Cinética , Microscopia Eletrônica/métodos , Modelos Biológicos , Proteínas Motores Moleculares/metabolismo , Movimento (Física) , Subfragmentos de Miosina/metabolismo , Pinças Ópticas , Estrutura Terciária de ProteínaRESUMO
The Mus musculus myosin-18A gene is expressed as two alternatively spliced isoforms, α and ß, with reported roles in Golgi localization, in maintenance of cytoskeleton, and as receptors for immunological surfactant proteins. Both myosin-18A isoforms feature a myosin motor domain, a single predicted IQ motif, and a long coiled-coil reminiscent of myosin-2. The myosin-18Aα isoform, additionally, has an N-terminal PDZ domain. Recombinant heavy meromyosin- and subfragment-1 (S1)-like constructs for both myosin-18Aα and -18ß species were purified from the baculovirus/Sf9 cell expression system. These constructs bound both essential and regulatory light chains, indicating an additional noncanonical light chain binding site in the neck. Myosin-18Aα-S1 and -18Aß-S1 molecules bound actin weakly with Kd values of 4.9 and 54 µm, respectively. The actin binding data could be modeled by assuming an equilibrium between two myosin conformations, a competent and an incompetent form to bind actin. Actin binding was unchanged by presence of nucleotide. Both myosin-18A isoforms bound N-methylanthraniloyl-nucleotides, but the rate of ATP hydrolysis was very slow (<0.002 s(-1)) and not significantly enhanced by actin. Phosphorylation of the regulatory light chain had no effect on ATP hydrolysis, and neither did the addition of tropomyosin or of GOLPH3, a myosin-18A binding partner. Electron microscopy of myosin-18A-S1 showed that the lever is strongly angled with respect to the long axis of the motor domain, suggesting a pre-power stroke conformation regardless of the presence of ATP. These data lead us to conclude that myosin-18A does not operate as a traditional molecular motor in cells.
Assuntos
Miosinas/química , Actinas/metabolismo , Trifosfato de Adenosina/química , Animais , Baculoviridae/metabolismo , Movimento Celular , Clonagem Molecular , Humanos , Hidrólise , Cinética , Luz , Camundongos , Microscopia Eletrônica/métodos , Modelos Moleculares , Músculo Esquelético/metabolismo , Miosinas/metabolismo , Fosforilação , Ligação Proteica , Isoformas de Proteínas , Estrutura Terciária de Proteína , Coelhos , Tropomiosina/químicaRESUMO
Homologous recombination (HR) is a key process in the repair of double-stranded DNA breaks (DSBs) that can initiate cancer or cell death. Human Bloom's syndrome RecQ-family DNA helicase (BLM) exerts complex activities to promote DSB repair while avoiding illegitimate HR. The oligomeric assembly state of BLM has been a key unresolved aspect of its activities. In this study we assessed the structure and oligomeric state of BLM, in the absence and presence of key HR-intermediate DNA structures, by using single-molecule visualization (electron microscopic and atomic force microscopic single-particle analysis) and solution biophysical (dynamic light scattering, kinetic and equilibrium binding) techniques. Besides full-length BLM, we used a previously characterized truncated construct (BLM(642-1290)) as a monomeric control. Contrary to previous models proposing a ring-forming oligomer, we found the majority of BLM molecules to be monomeric in all examined conditions. However, BLM showed a tendency to form dimers when bound to branched HR intermediates. Our results suggest that HR activities requiring single-stranded DNA translocation are performed by monomeric BLM, while complex DNA structures encountered and dissolved by BLM in later stages of HR induce partial oligomerization of the helicase.
Assuntos
DNA de Cadeia Simples/metabolismo , Recombinação Homóloga , RecQ Helicases/química , Trifosfato de Adenosina/metabolismo , Sequência de Aminoácidos , DNA de Cadeia Simples/química , Humanos , Hidrólise , Dados de Sequência Molecular , Ligação Proteica , Multimerização Proteica , RecQ Helicases/metabolismoRESUMO
Fluorescent nanodiamonds (FNDs) emit in the near-IR and do not photobleach or photoblink. These properties make FNDs better suited for numerous imaging applications compared with commonly used fluorescence agents such as organic dyes and quantum dots. However, nanodiamonds do not form stable suspensions in aqueous buffer, are prone to aggregation, and are difficult to functionalize. Here we present a method for encapsulating nanodiamonds with silica using an innovative liposome-based encapsulation process that renders the particle surface biocompatible, stable, and readily functionalized through routine linking chemistries. Furthermore, the method selects for a desired particle size and produces a monodisperse agent. We attached biotin to the silica-coated FNDs and tracked the three-dimensional motion of a biotinylated FND tethered by a single DNA molecule with high spatial and temporal resolution.
Assuntos
Coloides/química , Nanodiamantes , Dióxido de Silício/química , DNA/química , Fluorescência , Lipossomos , Espectroscopia de Luz Próxima ao Infravermelho , Propriedades de SuperfícieRESUMO
The powerstroke of the myosin motor is the basis of cell division and bodily movement, but has eluded empirical description due to the short lifetime and low abundance of intermediates during force generation. To gain insight into this process, we used well-established single-tryptophan and pyrene fluorescent sensors and electron microscopy to characterize the structural and kinetic properties of myosin complexed with ADP and blebbistatin, a widely used inhibitor. We found that blebbistatin does not weaken the tight actin binding of myosin.ADP, but unexpectedly it induces lever priming, a process for which the gamma-phosphate of ATP (or its analog) had been thought necessary. The results indicate that a significant fraction of the myosin.ADP.blebbistatin complex populates a previously inaccessible conformation of myosin resembling the start of the powerstroke.
Assuntos
Difosfato de Adenosina/química , Dictyostelium/metabolismo , Compostos Heterocíclicos de 4 ou mais Anéis/química , Miosinas/química , Trifosfato de Adenosina/química , Animais , Sítios de Ligação , Corantes Fluorescentes/química , Cinética , Microscopia Eletrônica/métodos , Microscopia de Fluorescência/métodos , Modelos Biológicos , Conformação Molecular , Conformação Proteica , CoelhosRESUMO
Septins are a family of conserved filament-forming proteins that function in multiple cellular processes. The number of septin genes within an organism varies, and higher eukaryotes express many septin isoforms due to alternative splicing. It is unclear if different combinations of septin proteins in complex alter the polymers' biophysical properties. We report that a duplication event within the CDC11 locus in Ashbya gossypii gave rise to two similar but distinct Cdc11 proteins: Cdc11a and Cdc1b. CDC11b transcription is developmentally regulated, producing different amounts of Cdc11a- and Cdc11b-complexes in the lifecycle of Ashbya gossypii. Deletion of either gene results in distinct cell polarity defects, suggesting non-overlapping functions. Cdc11a and Cdc11b complexes have differences in filament length and membrane-binding ability. Thus, septin subunit composition has functional consequences on filament properties and cell morphogenesis. Small sequence differences elicit distinct biophysical properties and cell functions of septins, illuminating how gene duplication could be a driving force for septin gene expansions seen throughout the tree of life.