Showing papers by "Randall Division of Cell and Molecular Biophysics published in 2006"

The Skeletal Muscle Satellite Cell: The Stem Cell That Came in From the Cold:

Abstract: The muscle satellite cell was first described and actually named on the basis of its anatomic location under the basement membrane surrounding each myofiber. For many years following its discovery, electron microscopy provided the only definitive method of identification. More recently, several molecular markers have been described that can be used to detect satellite cells, making them more accessible for study at the light microscope level. Satellite cells supply myonuclei to growing myofibers before becoming mitotically quiescent in muscle as it matures. They are then activated from this quiescent state to fulfill their roles in routine maintenance, hypertrophy, and repair of adult muscle. Because muscle is able to efficiently regenerate after repeated bouts of damage, systems must be in place to maintain a viable satellite cell pool, and it was proposed over 30 years ago that self-renewal was the primary mechanism. Self-renewal entails either a stochastic event or an asymmetrical cell division, where one daughter cell is committed to differentiation whereas the second continues to proliferate or becomes quiescent. This classic model of satellite cell self-renewal and the importance of satellite cells in muscle maintenance and repair have been challenged during the past few years as bone marrow-derived cells and various intramuscular populations were shown to be able to contribute myonuclei and occupy the satellite cell niche. This is a fast-moving and dynamic field, however, and in this review we discuss the evidence that we think puts this enigmatic cell firmly back at the center of adult myogenesis.

Pax7 and myogenic progression in skeletal muscle satellite cells.

Abstract: Skeletal muscle growth and regeneration are attributed to satellite cells - muscle stem cells resident beneath the basal lamina that surrounds each myofibre. Quiescent satellite cells express the transcription factor Pax7 and when activated, coexpress Pax7 with MyoD. Most then proliferate, downregulate Pax7 and differentiate. By contrast, others maintain Pax7 but lose MyoD and return to a state resembling quiescence. Here we show that Pax7 is able to drive transcription in quiescent and activated satellite cells, and continues to do so in those cells that subsequently cease proliferation and withdraw from immediate differentiation. We found that constitutive expression of Pax7 in satellite-cell-derived myoblasts did not affect MyoD expression or proliferation. Although maintained expression of Pax7 delayed the onset of myogenin expression it did not prevent, and was compatible with, myogenic differentiation. Constitutive Pax7 expression in a Pax7-null C2C12 subclone increased the proportion of cells expressing MyoD, showing that Pax7 can act genetically upstream of MyoD. However these Pax7-null cells were unable to differentiate into normal myotubes in the presence of Pax7. Therefore Pax7 may be involved in maintaining proliferation and preventing precocious differentiation, but does not promote quiescence.

The inner nuclear membrane protein emerin regulates beta-catenin activity by restricting its accumulation in the nucleus.

Abstract: Emerin is a type II inner nuclear membrane (INM) protein of unknown function. Emerin function is likely to be important because, when it is mutated, emerin promotes both skeletal muscle and heart defects. Here we show that one function of Emerin is to regulate the flux of β‐catenin, an important transcription coactivator, into the nucleus. Emerin interacts with β‐catenin through a conserved adenomatous polyposis coli (APC)‐like domain. When GFP‐emerin is expressed in HEK293 cells, β‐catenin is restricted to the cytoplasm and β‐catenin activity is inhibited. In contrast, expression of an emerin mutant, lacking its APC‐like domain (GFP‐emerinΔ), dominantly stimulates β‐catenin activity and increases nuclear accumulation of β‐catenin. Human fibroblasts that are null for emerin have an autostimulatory growth phenotype. This unusual growth phenotype arises through enhanced nuclear accumulation and activity of β‐catenin and can be replicated in wild‐type fibroblasts by transfection with constitutively active β‐catenin. Our results support recent findings that suggest that INM proteins can influence signalling pathways by restricting access of transcription coactivators to the nucleus.

Randomised controlled trials of conventional antipsychotic versus new atypical drugs, and new atypical drugs versus clozapine, in people with schizophrenia responding poorly to, or intolerant of, current drug treatment.

Abstract: Objectives: To determine the clinical and costeffectiveness of different classes of antipsychotic drug treatment in people with schizophrenia responding inadequately to, or having unacceptable side-effects from, their current medication. Design: Two pragmatic, randomised controlled trials (RCTs) were undertaken. The first RCT (band 1) compared the class of older, inexpensive conventional drugs with the class of new atypical drugs in people with schizophrenic disorders, whose current antipsychotic drug treatment was being changed either because of inadequate clinical response or owing to side-effects. The second RCT (band 2) compared the new (non-clozapine) atypical drugs with clozapine in people whose medication was being changed because of poor clinical response to two or more antipsychotic drugs. Both RCTs were four-centre trials with concealed randomisation and three follow-up assessments over 1 year, blind to treatment. Setting: Adult mental health settings in England. Participants: In total, 227 participants aged 18?65 years (40% of the planned sample) were randomised to band 1 and 136 (98% of the planned sample) to band 2. Interventions: Participants were randomised to a class of drug. The managing clinician selected the individual drug within that class, except for the clozapine arm in band 2. The new atypical drugs included risperidone, olanzapine, quetiapine and amisulpride. The conventional drugs included older drugs, including depot preparations. As in routine practice, clinicians and participants were aware of the identity of the prescribed drug, but clinicians were asked to keep their participating patient on the randomised medication for at least the first 12 weeks. If the medication needed to be changed, the clinician was asked to prescribe another drug within the same class, if possible. Main outcome measures: The primary outcome was the Quality of Life Scale (QLS). Secondary clinical outcomes included symptoms [Positive and Negative Syndrome Scale (PANSS)], side-effects and participant satisfaction. Economic outcomes were costs of health and social care and a utility measure. Results: Recruitment to band 1 was less than anticipated (40%) and diminished over the trial. This appeared largely due to loss of perceived clinical equipoise (clinicians progressively becoming more convinced of the superiority of new atypicals). Good follow-up rates and a higher than expected correlation between QLS score at baseline and at follow-up meant that the sample as recruited had 75% power to detect a difference in QLS score of 5 points between the two treatment arms at 52 weeks. The recruitment to band 2 was approximately as planned. Follow-up assessments were completed at week 52 in 81% of band 1 and 87% of band 2 participants. Band 1 data showed that, on the QLS and symptom measures, those participants in the conventional arm tended towards greater improvements. This suggests that the failure to find the predicted advantage for new atypicals was not due to inadequate recruitment and statistical power in this sample. Participants reported no clear preference for either class of drug. There were no statistically significant differential outcomes for participants entering band 1 for reasons of treatment intolerance to those entering because of broadly defined treatment resistance. Net costs over the year varied widely, with a mean of �18,850 in the conventional drug group and �20,123 in the new atypical group, not a statistically significant difference. Of these costs, 2.1% and 3.8% were due to antipsychotic drug costs in the conventional and atypical group, respectively. There was a trend towards participants in the conventional drug group scoring more highly on the utility measure at 1 year. The results for band 2 showed an advantage for commencing clozapine in quality of life (QLS) at trend level (p = 0.08) and in symptoms (PANSS), which was statistically significant (p = 0.01), at 1 year. Clozapine showed approximately a 5-point advantage on PANSS total score and a trend towards having fewer total extrapyramidal side-effects. Participants reported at 12 weeks that their mental health was significantly better with clozapine than with new atypicals (p < 0.05). Net costs of care varied widely, but were higher than in band 1, with a mean of �33,800 in the clozapine group and �28,400 in the new atypical group. Of these costs, 4.0% and 3.3%, respectively, were due to antipsychotic drug costs. The increased costs in the clozapine group appeared to reflect the licensing requirement for inpatient admission for commencing the drug. There was a trend towards higher mean participant utility scores in the clozapine group. Conclusions: For band 1, there is no disadvantage in terms of quality of life and symptoms, or associated costs of care, over 1 year in commencing conventional antipsychotic drugs rather than new atypical drugs. Conventional drugs were associated with nonsignificantly better outcomes and lower costs. Drug costs represented a small proportion of the overall costs of care (<5%). For band 2, there is a statistically significant advantage in terms of symptoms but not quality of life over 1 year in commencing clozapine rather than new atypical drugs, but with increased associated costs of care. The results suggest that conventional antipsychotic drugs, which are substantially cheaper, still have a place in the treatment of patients unresponsive to, or intolerant of, current medication. Further analyses of this data set are planned and further research is recommended into areas such as current antipsychotic treatment guidance, valid measures of utility in serious mental illness, lowdose ?conventional? treatment in first episode schizophrenia, QLS validity and determinants of QLS score in schizophrenia, and into the possible financial and other mechanisms of rewarding clinician participation in trials.

A dark yellow fluorescent protein (YFP)-based Resonance Energy-Accepting Chromoprotein (REACh) for Förster resonance energy transfer with GFP.

Abstract: Forster resonance energy transfer (FRET) microscopy is a powerful technique that enables the visualization of signaling intermediates, protein interactions, and protein conformational and biochemical status. With the availability of an ever-increasing collection of fluorescent proteins, pairs of spectrally different variants have been used for the study of FRET in living cells. However, suitable spectral overlap, necessary for efficient FRET, is limited by the requirement for proper emission separation. Currently used FRET pairs represent compromises between these opposing spectral demands that reduce the maximally attainable FRET sensitivity. We present a previously undescribed FRET acceptor, a nonfluorescent yellow fluorescent protein (YFP) mutant called REACh (for Resonance Energy-Accepting Chromoprotein). REACh allows the use of the photophysically superior FRET donor EGFP, with which it exhibits optimal spectral overlap, which obviates the need for narrow spectral filtering and allows additional fluorescent labels to be used within the same cell. The latter allows the generation of sophisticated bioassays for complex biological questions. We show that this dark acceptor is ideally suited for donor fluorescence lifetime imaging microscopy (FLIM) and confirm these measurements with an independent intensity-based donor fluorescence quenching resonance energy transfer (FqRET) assay. REACh also can be used in donor photobleaching kinetics-based FRET studies. By detecting FRET between a GFP-tagged ubiquitination substrate and REACh-labeled ubiquitin, we imaged the active ubiquitination machinery inside cells. This assay therefore can be used to study proteins whose function is regulated by ubiquitination.

Silencing of EphA3 through a cis interaction with ephrinA5.

Abstract: EphAs and ephrinAs are expressed in multiple areas of the developing brain in overlapping countergradients, notably in the retina and tectum. Here they are involved in targeting retinal axons to their correct topographic position in the tectum. We have used truncated versions of EphA3, single-amino acid point mutants of ephrinA5 and fluorescence resonance energy transfer technology to uncover a cis interaction between EphA3 and ephrinA5 that is independent of the established ligand-binding domain of EphA3. This cis interaction abolishes the induction of tyrosine phosphorylation of EphA3 and results in a loss of sensitivity of retinal axons to ephrinAs in trans. Our data suggest that formation of this complex transforms the uniform expression of EphAs in the nasal part of the retina into a gradient of functional EphAs and has a key role in controlling retinotectal mapping.

Palindromic assembly of the giant muscle protein titin in the sarcomeric Z-disk

Abstract: The Z-disk of striated and cardiac muscle sarcomeres is one of the most densely packed cellular structures in eukaryotic cells. It provides the architectural framework for assembling and anchoring the largest known muscle filament systems by an extensive network of protein-protein interactions, requiring an extraordinary level of mechanical stability. Here we show, using X-ray crystallography, how the amino terminus of the longest filament component, the giant muscle protein titin, is assembled into an antiparallel (2:1) sandwich complex by the Z-disk ligand telethonin. The pseudosymmetric structure of telethonin mediates a unique palindromic arrangement of two titin filaments, a type of molecular assembly previously found only in protein-DNA complexes. We have confirmed its unique architecture in vivo by protein complementation assays, and in vitro by experiments using fluorescence resonance energy transfer. The model proposed may provide a molecular paradigm of how major sarcomeric filaments are crosslinked, anchored and aligned within complex cytoskeletal networks.

Imaging macrophage chemotaxis in vivo: Studies of microtubule function in zebrafish wound inflammation

Abstract: The inflammatory response is one of the most dramatic examples of directed cell movement in nature. Inflammation is triggered at the site of injury and results in the migration of immune cells to the site to protect the host from infection. We have devised an in vivo inflammation assay using translucent zebrafish embryos, which allow live imaging and pharmacological manipulation of macrophage chemotaxis to wounds inflicted with a laser. Using this assay, we test the role of the microtubule cytoskeleton in macrophage chemotaxis in vivo using nocodazole to disrupt microtubule polymerization. We find that de-stabilisation of microtubules with nocodazole impairs macrophage recruitment to wounds, but that addition of the Rho kinase inhibitor Y-27632 suppresses these effects and restores the recruitment of macrophages to wounds. Taken together, these results suggest that destabilizing microtubules activates Rho kinase and that this increase in Rho kinase activity interferes with leukocyte recruitment in vivo.

Breaking the resolution limit in light microscopy

Abstract: Fluorescent imaging microscopy has been an essential tool for biologists over many years, especially after the discovery of the green fluorescent protein and the possibility of tagging virtually every protein with it. In recent years dramatic enhancement of the level of detail at which a fluorescing structure of interest can be imaged have been achieved. We review classical and new developments in high-resolution microscopy, and describe how these methods have been used in biological research. Classical methods include widefield and confocal microscopy whereas novel approaches range from linear methods such as 4Pi, I(5) and structured illumination microscopy to non-linear schemes such as stimulated emission depletion and saturated structured illumination. Localization based approaches (e.g. PALM and STORM), near-field methods and total internal refraction microscopy are also discussed. As the terms 'resolution', 'sensitivity', 'sampling' and 'precision' are sometimes confused, we explain their clear distinction. Key concepts such as the point spread function and the Abbe limit, which are necessary for an in depth understanding of the presented methods, are described without requiring extensive mathematical training.

Entry of muscle satellite cells into the cell cycle requires sphingolipid signaling.

Abstract: Adult skeletal muscle is able to repeatedly regenerate because of the presence of satellite cells, a population of stem cells resident beneath the basal lamina that surrounds each myofiber. Little is known, however, of the signaling pathways involved in the activation of satellite cells from quiescence to proliferation, a crucial step in muscle regeneration. We show that sphingosine-1-phosphate induces satellite cells to enter the cell cycle. Indeed, inhibiting the sphingolipid-signaling cascade that generates sphingosine-1-phosphate significantly reduces the number of satellite cells able to proliferate in response to mitogen stimulation in vitro and perturbs muscle regeneration in vivo. In addition, metabolism of sphingomyelin located in the inner leaflet of the plasma membrane is probably the main source of sphingosine-1-phosphate used to mediate the mitogenic signal. Together, our observations show that sphingolipid signaling is involved in the induction of proliferation in an adult stem cell and a key component of muscle regeneration.

Estrogen receptor alpha up-regulation and redistribution in human heart failure

Abstract: Clinical and animal studies suggest that estrogen receptors are involved in the development of myocardial hypertrophy and heart failure. In this study, we investigated whether human myocardial estr...

Rho GTPases and Leukocyte Adhesion Receptor Expression and Function in Endothelial Cells

Abstract: Rho family GTPases are key signal transducers that regulate cell adhesion and migration and a variety of other cellular responses, including changes in gene expression. In this review, we discuss how Rho GTPases regulate signaling by endothelial cell receptors involved in leukocyte extravasation. First, Rho GTPases affect the expression of some leukocyte adhesion molecules on endothelial cells, such as intracellular adhesion molecule-1 and E-selectin, that can be induced by proinflammatory mediators, hypoxia, or shear stress. Second, Rho GTPases are activated by engagement of several leukocyte adhesion receptors and contribute to both early morphological changes and subsequent alterations in gene expression. Rho GTPases are therefore candidate targets for inhibiting leukocyte transendothelial migration in heart disease and chronic inflammatory disorders.

Differential regulation of phagosome maturation in macrophages and dendritic cells mediated by Rho GTPases and ezrin–radixin–moesin (ERM) proteins

Abstract: Deletion of apoptotic cells from tissues involves their phagocytosis by macrophages, dendritic cells, and tissue cells. Although much attention has been focused on the participating ligands, receptors, and mechanisms of uptake, little is known of the disposition of the ingested cell within the phagosome. Here we show that uptake of apoptotic cells by macrophages or fibroblasts results in rapid phagosome maturation, whereas macrophage phagosomes containing Ig-opsonized target cells mature at a slower rate. The early maturation was shown to depend on activation of Rho acting through Rho kinase on ezrin–radixin–moesin proteins. Blockade of Rho signaling or inhibition of moesin both delayed maturation rates to those seen with opsonized targets. By contrast, phagosome maturation in dendritic cells was slower, similar between apoptotic and opsonized target cells, and unaffected by Rho inhibition. These observations have direct implications for the clearance of dying cells and the roles played by different phagocytes in antigen digestion and presentation.

PTEN couples Sema3A signalling to growth cone collapse.

Abstract: Distinct changes in glycogen synthase kinase-3 (GSK-3) signalling can regulate neuronal morphogenesis including the determination and maintenance of axonal identity, and are required for neurotrophin-mediated axon elongation. In addition, we have previously shown a dependency on GSK-3 activation in the semaphorin 3A (Sema3A)-mediated growth-cone-collapse response of sensory neurons. Regulation of GSK-3 activity involves the intermediate signalling lipid phosphatidylinositol 3,4,5-trisphosphate, which can be modulated by phosphatidylinositol 3-kinase (PI3K) and the tumour suppressor PTEN. We report here the involvement of PTEN in the Sema3A-mediated growth cone collapse. Sema3A suppresses PI3K signalling concomitant with the activation of GSK-3, which depends on the phosphatase activity of PTEN. PTEN is highly enriched in the axonal compartment and the central domain of sensory growth cones during axonal extension, where it colocalises with microtubules. Following exposure to Sema3A, PTEN accumulates rapidly at the growth cone membrane suggesting a mechanism by which PTEN couples Sema3A signalling to growth cone collapse. These findings demonstrate a dependency on PTEN to regulate GSK-3 signalling in response to Sema3A and highlight the importance of subcellular distributions of PTEN to control growth cone behaviour.

Inhibition of calpain stabilises podosomes and impairs dendritic cell motility.

Abstract: Podosomes, highly dynamic adhesion structures implicated in cell motility and extracellular matrix degradation, are characteristic of certain cells of the myeloid lineage and a limited range of other cell types. The nature and the mechanisms that regulate their high turnover are unknown at present. The cysteine protease calpain is involved in the regulation of cell migration in part by promoting either formation or disassembly of adhesion sites. Despite the fact that many known substrates of calpain are also structural components of the podosome complex, no studies have yet demonstrated that calpain participates in the regulation of podosome dynamics. In the present work, we show that inhibition of calpain in primary mouse dendritic cells leads to enhanced accumulation of actin filaments, the Wiskott Aldrich Syndrome protein (WASP), β 2 integrins, talin, paxillin and vinculin in podosomes. This accumulation of components is associated with stabilisation of podosome turnover, overall reduction in velocity of cell locomotion and impaired transmigration across an endothelial monolayer. We also demonstrate that calpain cleaves the podosome components talin, Pyk2 and WASP in dendritic cells. In summary, our results provide evidence that calpain regulates podosome composition and turnover and that this process is required for efficient migration of dendritic cells.

Electric field driven jetting: an emerging approach for processing living cells.

Abstract: This paper reports for the first time the ability to process living cellular materials by means of electrified jets at electric field strengths of up to 2 kV/mm. Bio-suspensions containing living human Jurkat cells at different concentrations were processed via this jetting approach. The jetting process was carried out at an electric field strength between 0.67 kV/mm and 2 kV/mm, corresponding to an applied voltage of 10-30 kV between two electrodes approximately 15 mm apart. The Jurkat cells were jetted under sterile conditions, collected in petri dishes and incubated for 24 and 48 hours. During and after incubation, cells were assessed for survival and structural damage; cells were found to be unharmed and to retain their integrity under all electric field strengths examined. At all field strengths jetting took place in the unstable mode. Good correlation was observed between droplet distribution plots generated by way of laser spectroscopy and estimated values from measurements of droplet relics.

The Transitional Junction: A New Functional Subcellular Domain at the Intercalated Disc

Abstract: We define here a previously unrecognized structural element close to the heart muscle plasma membrane at the intercalated disc where the myofibrils lead into the adherens junction. At this location, the plasma membrane is extensively folded. Immunofluorescence and immunogold electron microscopy reveal a spectrin-rich domain at the apex of the folds. These domains occur at the axial level of what would be the final Z-disc of the terminal sarcomere in the myofibril, although there is no Z-disc-like structure there. However, a sharp transitional boundary lies between the myofibrillar I-band and intercalated disc thin filaments, identifiable by the presence of Z-disc proteins, alpha-actinin, and N-terminal titin. This allows for the usual elastic positioning of the A-band in the final sarcomere, whereas the transduction of the contractile force normally associated with the Z-disc is transferred to the adherens junctions at the plasma membrane. The axial conjunction of the transitional junction with the spectrin-rich domains suggests a mechanism for direct communication between intercalated disc and contractile apparatus. In particular, it provides a means for sarcomeres to be added to the ends of the cells during growth. This is of particular relevance to understanding myocyte elongation in dilated cardiomyopathy.

A bioinformatician's view of the metabolome.

Abstract: The study of a collection of metabolites as a whole (metabolome), as opposed to isolated small molecules, is a fast-growing field promising to take us one step further towards understanding cell biology, and relating the genetic capabilities of an organism to its observed phenotype. The new sciences of metabolomics and metabonomics can exploit a variety of existing experimental and computational methods, but they also require new technology that can deal with both the amount and the diversity of the data relating to the rich world of metabolites. More specifically, the collaboration between bioinformaticians and chemoinformaticians promises to advance our view of cognate molecules, by shedding light on their atomic structure and properties. Modelling of the interactions of metabolites with other entities in the cell, and eventually complete modelling of reaction pathways will be essential for analysis of the experimental data, and prediction of an organism's response to environmental challenges.

Electrohydrodynamic jetting of mouse neuronal cells.

Abstract: CAD (Cath.a-differentiated) cells, a mouse neuronal cell line, were subjected to electrohydrodynamic jetting at a field strength of 0.47–0.67 kV/mm, corresponding to an applied voltage of 7–10 kV. After jetting, the cells appeared normal and continued to divide at rates similar to those shown by control samples. Jetted cells, when placed in serum-free medium, underwent differentiation that was sustained for at least 1 month. Some of the droplets produced by jetting contained only a few cells. These results indicate that the process of jetting does not significantly perturb neuronal cells and that this novel approach might in the future be a useful way to deposit small numbers of living nerve cells on to surfaces.

Sphingomyelin Levels in the Plasma Membrane Correlate with the Activation State of Muscle Satellite Cells

Abstract: Satellite cells are responsible for postnatal growth, hypertrophy, and regeneration of skeletal muscle. They are normally quiescent, and must be activated to fulfill these functions, yet little is known of how this is regulated. As a first step in determining the role of lipids in this process, we examined the dynamics of sphingomyelin in the plasma membrane. Sphingomyelin contributes to caveolae/lipid rafts, which act to concentrate signaling molecules, and is also a precursor of several bioactive lipids. Proliferating or differentiated C2C12 muscle cells did not bind lysenin, a sphingomyelin-specific binding protein, but noncycling reserve cells did. Quiescent satellite cells also bound lysenin, revealing high levels of sphingomyelin in their plasma membranes. On activation, however, the levels of sphingomyelin drop, so that lysenin did not label proliferating satellite cells. Although most satellite cell progeny differentiate, others stop cycling, maintain Pax7, downregulate MyoD, and escape immediate differentiation. Importantly, many of these Pax7-positive/MyoD-negative cells also regained lysenin binding on their surface, showing that the levels of sphingomyelin had again increased. Our observations show that quiescent satellite cells are characterized by high levels of sphingomyelin in their plasma membranes and that lysenin provides a novel marker of myogenic quiescence.

Structural changes in the myosin filament and cross-bridges during active force development in single intact frog muscle fibres: stiffness and X-ray diffraction measurements.

Abstract: Structural and mechanical changes occurring in the myosin filament and myosin head domains during the development of the isometric tetanus have been investigated in intact frog muscle fibres at 4°C and 2.15 μm sarcomere length, using sarcomere level mechanics and X-ray diffraction at beamline ID2 of the European Synchrotron Radiation Facility (Grenoble, France). The time courses of changes in both the M3 and M6 myosin-based reflections were recorded with 5 ms frames using the gas-filled RAPID detector (MicroGap Technology). Following the end of the latent period (11 ms after the start of stimulation), force increases to the tetanus plateau value (T0) with a half-time of 40 ms, and the spacings of the M3 and M6 reflections (SM3 and SM6) increase by 1.5% from their resting values, with time courses that lead that of force by ∼10 and ∼20 ms, respectively. These temporal relations are maintained when the increase of force is delayed by ∼10 ms by imposing, from 5 ms after the first stimulus, 50 nm (half-sarcomere)−1 shortening at the velocity (V0) that maintains zero force. Shortening at V0 transiently reduces SM3 following the latent period and delays the subsequent increase in SM3, but only delays the SM6 increase without a transient decrease. Shortening at V0 imposed at the tetanus plateau causes an abrupt reduction of the intensity of the M3 reflection (IM3), whereas the intensity of the M6 reflection (IM6) is only slightly reduced. The changes in half-sarcomere stiffness indicate that the isometric force at each time point is proportional to the number of myosin heads bound to actin. The different sensitivities of the intensity and spacing of the M3 and M6 reflections to the mechanical responses support the view that the M3 reflection in active muscle originates mainly from the myosin heads attached to the actin filament and the M6 reflection originates mainly from a fixed structure in the myosin filament signalling myosin filament length changes during the tetanus rise.