Pollen tube extract supports the movement of actin filaments in vitro

Summary Muscle actin filaments labeled with rhodamine-phalloidin were observed to move on the surface coated with a crude extract of pollen tubes ofLilium longiflorum with an average velocity of 1.99±0.55 m/sec. The movement required both Mg²⁺ and ATP. These results indicate that the extract of pollen tubes contains a myosin-like translocatorAbbreviations ATP adenosine-5-triphosphate - DTT dithiothreitol - EGTA ethyleneglycol-bis-(-aminoethylether)N,N,N,N-tetraacetic acid - PIPES piperazine-N,N-bis-(2-ethanesulfonic acid) - PMSF phenylmethylsulfonyl fluoride     

洋葱鳞茎中肌球蛋白和肌动蛋白的免疫化学鉴定

制备了粘菌肌动蛋白抗血清和鸡骨骼肌肌球蛋白抗血清。用这两种抗血清,经对流免疫电泳,火箭电泳及酶联免疫吸附分析,证明了高等植物洋葱鳞茎中肌球蛋白和肌动蛋白的存在。     

Communicative interaction of myosins along an actin filament in the presence of ATP

Myosin molecules contacting an actin filament in the presence of ATP were found to regulate the filamental fluctuations due to ATP hydrolysis in a communicative manner along the filament. As an evidence of the occurrence of the communication, ATP-activated fluctuating displacements of the filament in the direction perpendicular to its longitudinal axis were identified to propagate at a finite velocity not less than about 0.2 μm/s unidirectionally along the filament.     

Distribution of actin and myosin in muscle and non-muscle cells

Summary Specific anti-actin and anti-myosin antibodies were shown to react in single and double immunofluorescence sandwich tests with identical sites in non-muscle cells in frozen sections of tissues and in cultured cells. In tissues, both antibodies reacted with liver cell membranes, parts of renal glomeruli, brush borders and peritubular fibrils of renal tubules, brain synaptic junctions, and membranes of lymphoid cells in thymic medulla, lymph nodes and spleen. Both antibodies reacted strongly with long parallel cytoplasmic fibrils in cultured fibroblasts, and with disrupted fibrils in cytochalasin-B treated cells. In neuroblastoma cells both antibodies gave prominent staining of growth cones and microspikes. The observation that the distribution of myosin parallels that of actin in non-muscle cells argues strongly in favour of a functional interaction between the two molecules in the generation of contractile activity in nonmuscle cells.The authors thank Dr. M. Owen, National Institute of Medical Research, Mill Hill, for the gift of rabbit anti-actin antibodyOn sabbatical leave from Monash University, and supported by a Commonwealth Medical FellowshipThe Brompton Hospital, London     

Staggered movement of an actin filament sliding on myosin molecules in the presence of ATP

An actin filament sliding on myosin molecules in the presence of an extremely low concentration of ATP exhibited a staggered movement. Longitudinally sliding movement of the filament was frequently interrupted by its non-sliding, fluctuating movements both in the longitudinal and transversal directions. Intermittent sliding movements of an actin filament indicate establishment of a coordination of ATP-mediated active sites distributed along the filament.     

Calyculin A-induced neurite retraction is critically dependent on actomyosin activation but not on polymerization state of microtubules

Calyculin A (CL-A), a toxin isolated from the marine sponge Discodermia calyx, is a strong inhibitor of protein phosphatase 1 (PP1) and 2A (PP2A). Although CL-A is known to induce rapid neurite retraction in developing neurons, the cytoskeletal dynamics of this retraction have remained unclear. Here, we investigated the cytoskeletal dynamics during CL-A-induced neurite retraction in cultured rat hippocampal neurons, using fluorescence microscopy as well as polarized light microscopy, which can visualize the polymerization state of the cytoskeleton in living cells. We observed that MTs were bent while maintaining their polymerization state during the neurite retraction. In addition, we also found that CL-A still induced neurite retraction when MTs were depolymerized by nocodazole or stabilized by paclitaxel. These results imply a mechanism other than depolymerization of MTs for CL-A-induced neurite retraction. Our pharmacological studies showed that blebbistatin and cytochalasin D, an inhibitor of myosin II and a depolymerizer of actin, strongly inhibited CL-A-induced neurite retraction. Based on all these findings, we propose that CL-A generates strong contractile forces by actomyosin to induce rapid neurite retraction independently from MT depolymerization.     

The contractile apparatus of striated muscle in the course of atrophy and regeneration

Summary Changes in the contractile apparatus of denervated rat soleus muscles were investigated during the course of reinnervation.As observed earlier, in the course of denervation atrophy the ratio of myosin to actin filaments decreases because myosin filaments disappear faster than actin filaments (Jakubiec-Puka et al. 1981 a). After reinnervation the amount of myosin filaments and myosin heavy chains (myosin HC) in the muscle increased during the first few days; the increment of actin content was negligible. The proportion of myosin HC to actin remained lower than normal for about 30 days. The excess of actin filaments frequently observed in the newly-formed myofibrils reflects this disproportion.The results show a lability of myosin and suggest some cytoskeletal role for actin filaments.     

The contractile apparatus of striated muscle in the course of atrophy and regeneration

Summary The ultrastructure of the contractile apparatus of the rat soleus muscle during the course of denervation atrophy was investigated. It was found that the ratio of thin to thick filaments increased in myofibrils of atrophying muscle fibers. Elevation of the ratio was observed as early as the second day after denervation, and became more pronounced with the progress of atrophy. Parallel measurements of the amounts of actin and myosin in the myofibrils and in the muscle protein extracts revealed a lower proportion of myosin heavy chains to actin in the fractions from denervated muscles, compared with the control values. Both the electron-microscopic observations and the biochemical evaluation of the actin content of the muscle, suggests that the elevated ratio of thin to thick filaments seen in the course of the muscle atrophy appears as the result of an earlier and more intensive disappearance of thick filaments. Thin filaments disappeared more slowly, in parallel to the decrease in muscle weight.On the basis of the results presented a mechanism of progress of simple atrophy of muscle in suggested.     

Cardiac Muscle Activation Blunted by a Mutation to the Regulatory Component,Troponin T

The striated muscle thin filament comprises actin, tropomyosin, and troponin. The Tn complex consists of three subunits, troponin C (TnC), troponin I (TnI), and troponin T (TnT). TnT may serve as a bridge between the Ca²⁺ sensor (TnC) and the actin filament. In the short helix preceding the IT-arm region, H1(T2), there are known dilated cardiomyopathy-linked mutations (among them R205L). Thus we hypothesized that there is an element in this short helix that plays an important role in regulating the muscle contraction, especially in Ca²⁺ activation. We mutated Arg-205 and several other amino acid residues within and near the H1(T2) helix. Utilizing an alanine replacement method to compare the effects of the mutations, the biochemical and mechanical impact on the actomyosin interaction was assessed by solution ATPase activity assay, an in vitro motility assay, and Ca²⁺ binding measurements. Ca²⁺ activation was markedly impaired by a point mutation of the highly conserved basic residue R205A, residing in the short helix H1(T2) of cTnT, whereas the mutations to nearby residues exhibited little effect on function. Interestingly, rigor activation was unchanged between the wild type and R205A TnT. In addition to the reduction in Ca²⁺ sensitivity observed in Ca²⁺ binding to the thin filament, myosin S1-ADP binding to the thin filament was significantly affected by the same mutation, which was also supported by a series of S1 concentration-dependent ATPase assays. These suggest that the R205A mutation alters function through reduction in the nature of cooperative binding of S1.     

Force generation and shift of mass between myosin and actinin skinned striated muscle fibres at low calcium concentrations

Skinned muscle fibres from the gracilis muscle of the rabbit were used to record small angle X-ray diffraction spectra under various contractile conditions. The intracellular calcium concentration, expressed as pCa, was varied between 8.0 and 5.74. Equatorial diffraction spectra were fitted by a function consisting of five Gaussian curves and a hyperbola to separate the (1.0), (1.1), (2.0), (2.1) and Z-line diffraction peaks. The hyperbola was used to correct for residual scattering in the preparation. The ratio between the intensities of the (1.1) and (1.0) peaks was defined as the relative transfer of mass between myosin and actin, due to crossbridge formation after activation by calcium. The relation between the ratio and the relative force of the fibre (normalized to the force at pCa 5.74 and sarcomere length 2.0 μm) was linear. At high pCa (from pCa 6.34 to 8.0) no active force was observed, while the ratio still decreased. Sarcomere length was recorded by laser diffraction. The laser diffraction patterns did not show changes in sarcomere length due to activation in the high pCa range (between 8.0 and 6.34). From these results the conclusion is drawn that crossbridge movement occurs even at subthreshold calcium concentrations in the cell, when no active force is exerted. Since no force is generated this movement may be related to crossbridges in the weakly bound state. Received: 20 June 1996 / Revised version: 12 January 1998 / Accepted: 18 March 1998     

Localization of Myosin 1b to Actin Protrusions Requires Phosphoinositide Binding

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 organelles such as endosomes and lysosomes. Whether Myo1b directly associates with membranes is unknown. We demonstrate here that full-length rat Myo1b binds specifically and with high affinity to phosphatidylinositol 4,5-bisphosphate (PIP₂) and phosphatidylinositol 3,4,5-triphosphate (PIP₃), two phosphoinositides that play important roles in cell signaling. Binding is not Ca²⁺-dependent and does not involve the calmodulin-binding IQ region in the neck domain of Myo1b. Furthermore, the binding site is contained entirely within the C-terminal tail region, which contains a putative pleckstrin homology domain. Single mutations in the putative pleckstrin homology domain abolish binding of the tail domain of Myo1b to PIP₂ and PIP₃ in vitro. These same mutations alter the distribution of Myc-tagged Myo1b at membrane protrusions in HeLa cells where PIP₂ localizes. In addition, we found that motor activity is required for Myo1b localization in filopodia. These results suggest that binding of Myo1b to phosphoinositides plays an important role in vivo by regulating localization to actin-enriched membrane projections.     

肌球蛋白工作循环的机械化学偶联模型

目前在众多的分子马达中对骨骼肌肌球蛋白的研究较多,本文对肌球蛋白的结构、工作循环机制以及单分子动力学性质进行了探索。同时,对各种生化条件下肌纤维的收缩性质进行了测试。将Houdusse和Sweeney给出的机械化学偶联模型简化成一个新的四态模型,通过对定态时肌球蛋白态分布的研究,证明了简化模型的合理性。     

Specific Sorting and Post-Golgi Trafficking of Dendritic Potassium Channels in Living Neurons

Proper membrane localization of ion channels is essential for the function of neuronal cells. Particularly, the computational ability of dendrites depends on the localization of different ion channels in specific subcompartments. However, the molecular mechanisms that control ion channel localization in distinct dendritic subcompartments are largely unknown. Here, we developed a quantitative live cell imaging method to analyze protein sorting and post-Golgi vesicular trafficking. We focused on two dendritic voltage-gated potassium channels that exhibit distinct localizations: Kv2.1 in proximal dendrites and Kv4.2 in distal dendrites. Our results show that Kv2.1 and Kv4.2 channels are sorted into two distinct populations of vesicles at the Golgi apparatus. The targeting of Kv2.1 and Kv4.2 vesicles occurred by distinct mechanisms as evidenced by their requirement for specific peptide motifs, cytoskeletal elements, and motor proteins. By live cell and super-resolution imaging, we identified a novel trafficking machinery important for the localization of Kv2.1 channels. Particularly, we identified non-muscle myosin II as an important factor in Kv2.1 trafficking. These findings reveal that the sorting of ion channels at the Golgi apparatus and their subsequent trafficking by unique molecular mechanisms are crucial for their specific localizations within dendrites.     

Myosin 3A Kinase Activity Is Regulated by Phosphorylation of the Kinase Domain Activation Loop

Class III myosins are unique members of the myosin superfamily in that they contain both a motor and kinase domain. We have found that motor activity is decreased by autophosphorylation, although little is known about the regulation of the kinase domain. We demonstrate by mass spectrometry that Thr-178 and Thr-184 in the kinase domain activation loop and two threonines in the loop 2 region of the motor domain are autophosphorylated (Thr-908 and Thr-919). The kinase activity of MYO3A 2IQ with the phosphomimic (T184E) or phosphoblock (T184A) mutations demonstrates that kinase activity is reduced 30-fold as a result of the T184A mutation, although the Thr-178 site only had a minor impact on kinase activity. Interestingly, the actin-activated ATPase activity of MYO3A 2IQ is slightly reduced as a result of the T178A and T184A mutations suggesting coupling between motor and kinase domains. Full-length GFP-tagged T184A and T184E MYO3A constructs transfected into COS7 cells do not disrupt the ability of MYO3A to localize to filopodia structures. In addition, we demonstrate that T184E MYO3A reduces filopodia elongation in the presence of espin-1, whereas T184A enhances filopodia elongation in a similar fashion to kinase-dead MYO3A. Our results suggest that as MYO3A accumulates at the tips of actin protrusions, autophosphorylation of Thr-184 enhances kinase activity resulting in phosphorylation of the MYO3A motor and reducing motor activity. The differential regulation of the kinase and motor activities allows for MYO3A to precisely self-regulate its concentration in the actin bundle-based structures of cells.     

Mouse Myosin-19 Is a Plus-end-directed,High-duty Ratio Molecular Motor

Class XIX myosin (Myo19) is a vertebrate-specific unconventional myosin, responsible for the transport of mitochondria. To characterize biochemical properties of Myo19, we prepared recombinant mouse Myo19-truncated constructs containing the motor domain and the IQ motifs using the baculovirus/Sf9 expression system. We identified regulatory light chain (RLC) of smooth muscle/non-muscle myosin-2 as the light chain of Myo19. The actin-activated ATPase activity and the actin-gliding velocity of Myo19-truncated constructs were about one-third and one-sixth as those of myosin-5a, respectively. The apparent affinity of Myo19 to actin was about the same as that of myosin-5a. The RLCs bound to Myo19 could be phosphorylated by myosin light chain kinase, but this phosphorylation had little effect on the actin-activated ATPase activity and the actin-gliding activity of Myo19-truncated constructs. Using dual fluorescence-labeled actin filaments, we determined that Myo19 is a plus-end-directed molecular motor. We found that, similar to that of the high-duty ratio myosin, such as myosin-5a, ADP release rate was comparable with the maximal actin-activated ATPase activity of Myo19, indicating that ADP release is a rate-limiting step for the ATPase cycle of acto-Myo19. ADP strongly inhibited the actin-activated ATPase activity and actin-gliding activity of Myo19-truncated constructs. Based on the above results, we concluded that Myo19 is a high-duty ratio molecular motor moving to the plus-end of the actin filament.