The Dysferlin C2A Domain Binds PI(4,5)P2 and Penetrates Membranes.

Dysferlin is a large membrane protein found most prominently in striated muscle. Loss of dysferlin activity is associated with reduced exocytosis, abnormal intracellular Ca2+ and the muscle diseases limb-girdle muscular dystrophy and Miyoshi myopathy. The cytosolic region of dysferlin consists of seven C2 domains with mutations in the C2A domain at the N-terminus resulting in pathology. Despite the importance of Ca2+ and membrane binding activities of the C2A domain for dysferlin function, the mechanism of the domain remains poorly characterized. In this study we find that the C2A domain preferentially binds membranes containing PI(4,5)P2 through an interaction mediated by residues Y23, K32, K33, and R77 on the concave face of the domain. We also found that subsequent to membrane binding, the C2A domain inserts residues on the Ca2+ binding loops into the membrane. Analysis of solution NMR measurements indicate that the domain inhabits two distinct structural states, with Ca2+ shifting the population between states towards a more rigid structure with greater affinity for PI(4,5)P2. Based on our results, we propose a mechanism where Ca2+ converts C2A from a structurally dynamic, low PI(4,5)P2 affinity state to a high affinity state that targets dysferlin to PI(4,5)P2 enriched membranes through interaction with Tyr23, K32, K33, and R77. Binding also involves changes in lipid packing and insertion by the third Ca2+ binding loop of the C2 domain into the membrane, which would contribute to dysferlin function in exocytosis and Ca2+ regulation.

Orientation of the Dysferlin C2A Domain is Responsive to the Composition of Lipid Membranes.

Dysferlin is a 230 kD protein that plays a critical function in the active resealing of micron-sized injuries to the muscle sarcolemma by recruiting vesicles to patch the injured site via vesicle fusion. Muscular dystrophy is observed in humans when mutations disrupt this repair process or dysferlin is absent. While lipid binding by dysferlin's C2A domain (dysC2A) is considered fundamental to the membrane resealing process, the molecular mechanism of this interaction is not fully understood. By applying nonlinear surface-specific vibrational spectroscopy, we have successfully demonstrated that dysferlin's N-terminal C2A domain (dysC2A) alters its binding orientation in response to a membrane's lipid composition. These experiments reveal that dysC2A utilizes a generic electrostatic binding interaction to bind to most anionic lipid surfaces, inserting its calcium binding loops into the lipid surface while orienting its ß-sheets 30-40° from surface normal. However, at lipid surfaces, where PI(4,5)P2 is present, dysC2A tilts its ß-sheets more than 60° from surface normal to expose a polybasic face, while it binds to the PI(4,5)P2 surface. Both lipid binding mechanisms are shown to occur alongside dysC2A-induced lipid clustering. These different binding mechanisms suggest that dysC2A could provide a molecular cue to the larger dysferlin protein as to signal whether it is bound to the sarcolemma or another lipid surface.

Two Pathway-Specific Transcriptional Regulators, PltR and PltZ, Coordinate Autoinduction of Pyoluteorin in Pseudomonas protegens Pf-5.

Antibiotic biosynthesis by microorganisms is commonly regulated through autoinduction, which allows producers to quickly amplify the production of antibiotics in response to environmental cues. Antibiotic autoinduction generally involves one pathway-specific transcriptional regulator that perceives an antibiotic as a signal and then directly stimulates transcription of the antibiotic biosynthesis genes. Pyoluteorin is an autoregulated antibiotic produced by some Pseudomonas spp. including the soil bacterium Pseudomonas protegens Pf-5. In this study, we show that PltR, a known pathway-specific transcriptional activator of pyoluteorin biosynthesis genes, is necessary but not sufficient for pyoluteorin autoinduction in Pf-5. We found that pyoluteorin is perceived as an inducer by PltZ, a second pathway-specific transcriptional regulator that directly represses the expression of genes encoding a transporter in the pyoluteorin gene cluster. Mutation of pltZ abolished the autoinducing effect of pyoluteorin on the transcription of pyoluteorin biosynthesis genes. Overall, our results support an alternative mechanism of antibiotic autoinduction by which the two pathway-specific transcriptional regulators PltR and PltZ coordinate the autoinduction of pyoluteorin in Pf-5. Possible mechanisms by which PltR and PltZ mediate the autoinduction of pyoluteorin are discussed.

Truncation of the otoferlin transmembrane domain alters the development of hair cells and reduces membrane docking.

Release of neurotransmitter from sensory hair cells is regulated by otoferlin. Despite the importance of otoferlin in the auditory and vestibular pathways, the functional contributions of the domains of the protein have not been fully characterized. Using a zebrafish model, we investigated a mutant otoferlin with a stop codon at the start of the transmembrane domain. We found that both the phenotype severity and the expression level of mutant otoferlin changed with the age of the zebrafish. At the early developmental time point of 72 h post fertilization, low expression of the otoferlin mutant coincided with synaptic ribbon deficiencies, reduced endocytosis, and abnormal transcription of several hair cell genes. As development proceeded, expression of the mutant otoferlin increased, and both synaptic ribbons and hair cell transcript levels resembled wild type. However, hair cell endocytosis deficits and abnormalities in the expression of GABA receptors persisted even after up-regulation of mutant otoferlin. Analysis of membrane-reconstituted otoferlin measurements suggests a function for the transmembrane domain in liposome docking. We conclude that deletion of the transmembrane domain reduces membrane docking, attenuates endocytosis, and results in developmental delay of the hair cell.

Direct Evidence That Mutations within Dysferlin's C2A Domain Inhibit Lipid Clustering.

Mechanical stress on sarcolemma can create small tears in the muscle cell membrane. Within the sarcolemma resides the multidomain dysferlin protein. Mutations in this protein render it unable to repair the sarcolemma and have been linked to muscular dystrophy. A key step in dysferlin-regulated repair is the binding of the C2A domain to the lipid membrane upon increased intracellular calcium. Mutations mapped to this domain cause loss of binding ability of the C2A domain. There is a crucial need to understand the geometry of dysferlin C2A at a membrane interface as well as cell membrane lipid reorientation when compared to that of a mutant. Here, we describe a comparison between the wild-type dysferlin C2A and a mutation to the conserved aspartic acids in the domain binding loops. To identify both the geometry and the cell membrane lipid reorientation, we applied sum frequency generation (SFG) vibrational spectroscopy and coupled it with simulated SFG spectra to observe and quantify the interaction with a model cell membrane composed of phosphotidylserine and phosphotidylcholine. Observed changes in surface pressure demonstrate that calcium-bridged electrostatic interactions govern the initial interaction of the C2A domains docking with a lipid membrane. SFG spectra taken from the amide-I region for the wild type and variant contain features near 1642, 1663, and 1675 cm-1 related to the C2A domain ß-sandwich secondary structure, indicating that the domain binds in a specific orientation. Mapping simulated SFG spectra to the experimentally collected spectra indicated that both wild-type and variant domains have nearly the same orientation to the membrane surface. However, examining the ordering of the lipids that make up a model membrane using SFG, we find that the wild type clusters the lipids as seen by the increase in the ratio of the CD3 and CD2 symmetric intensities by 170% for the wild type and by 120% for the variant. This study highlights the capabilities of SFG to probe with great detail biological mutations in proteins at cell membrane interfaces.

Otoferlin Depletion Results in Abnormal Synaptic Ribbons and Altered Intracellular Calcium Levels in Zebrafish.

The protein otoferlin plays an essential role at the sensory hair cell synapse. Mutations in otoferlin result in deafness and depending on the species, mild to strong vestibular deficits. While studies in mouse models suggest a role for otoferlin in synaptic vesicle exocytosis and endocytosis, it is unclear whether these functions are conserved across species. To address this question, we characterized the impact of otoferlin depletion in zebrafish larvae and found defects in synaptic vesicle recycling, abnormal synaptic ribbons, and higher resting calcium concentrations in hair cells. We also observed abnormal expression of the calcium binding hair cell genes s100s and parvalbumin, as well as the nogo related proteins rtn4rl2a and rtn4rl2b. Exogenous otoferlin partially restored expression of genes affected by endogenous otoferlin depletion. Our results suggest that in addition to vesicle recycling, depletion of otoferlin disrupts resting calcium levels, alters synaptic ribbon architecture, and perturbs transcription of hair cells specific genes during zebrafish development.

Otoferlin C2F Domain-Induced Changes in Membrane Structure Observed by Sum Frequency Generation.

Proteins that contain C2 domains are involved in a variety of biological processes, including encoding of sound, cell signaling, and cell membrane repair. Of particular importance is the interface activity of the C-terminal C2F domain of otoferlin due to the pathological mutations known to significantly disrupt the protein's lipid membrane interface binding activity, resulting in hearing loss. Therefore, there is a critical need to define the geometry and positions of functionally important sites and structures at the otoferlin-lipid membrane interface. Here, we describe the first in situ probe of the protein orientation of otoferlin's C2F domain interacting with a cell membrane surface. To identify this protein's orientation at the lipid interface, we applied sum frequency generation (SFG) vibrational spectroscopy and coupled it with simulated SFG spectra to observe and quantify the otoferlin C2F domain interacting with model lipid membranes. A model cell membrane was built with equal amounts of phosphatidylserine and phosphatidylcholine. SFG measurements of the lipids that make up the model membrane indicate a 62% increase in amplitude from the SFG signal near 2075 cm-1 upon protein interaction, suggesting domain-induced changes in the orientation of the lipids and possible membrane curvature. This increase is related to lipid ordering caused by the docking interaction of the otoferlin C2F domain. SFG spectra taken from the amide-I region contain features near 1630 and 1670 cm-1 related to the C2F domains beta-sandwich secondary structure, thus indicating that the domain binds in a specific orientation. By mapping the simulated SFG spectra to the experimentally collected SFG spectra, we found the C2F domain of otoferlin orients 22° normal to the lipid surface. This information allows us to map what portion of the domain directly interacts with the lipid membrane.

Fer1l6 is essential for the development of vertebrate muscle tissue in zebrafish.

The precise spatial and temporal expression of genes is essential for proper organismal development. Despite their importance, however, many developmental genes have yet to be identified. We have determined that Fer1l6, a member of the ferlin family of genes, is a novel factor in zebrafish development. We find that Fer1l6 is expressed broadly in the trunk and head of zebrafish larvae and is more restricted to gills and female gonads in adult zebrafish. Using both genetic mutant and morpholino knockdown models, we found that loss of Fer1l6 led to deformation of striated muscle tissues, delayed development of the heart, and high morbidity. Further, expression of genes associated with muscle cell proliferation and differentiation were affected. Fer1l6 was also detected in the C2C12 cell line, and unlike other ferlin homologues, we found Fer1l6 expression was independent of the myoblast-to-myotube transition. Finally, analysis of cell and recombinant protein-based assays indicate that Fer1l6 colocalizes with syntaxin 4 and vinculin, and that the putative C2 domains interact with lipid membranes. We conclude that Fer1l6 has diverged from other vertebrate ferlins to play an essential role in zebrafish skeletal and cardiac muscle development.

Emerging Functional Differences between the Synaptotagmin and Ferlin Calcium Sensor Families.

The ferlin family proteins have emerged as multi-C2 domain regulators of calcium-triggered membrane fusion and fission events. While initially determined to share many of the features of members of the synaptotagmin family of calcium sensors, ferlins in more recent studies have been found to interact directly with non-neuronal voltage-gated calcium channels and nucleate the assembly of membrane-trafficking protein complexes, functions that distinguish them from the more well studied members of the synaptotagmin family. Here we highlight some of the recent findings that have advanced our understanding of ferlins and their functional differences with the synaptotagmin family.

Otoferlin is a multivalent calcium-sensitive scaffold linking SNAREs and calcium channels.

Sensory hair cells rely on otoferlin as the calcium sensor for exocytosis and encoding of sound preferentially over the neuronal calcium sensor synaptotagmin. Although it is established that synaptotagmin cannot rescue the otoferlin KO phenotype, the large size and low solubility of otoferlin have prohibited direct biochemical comparisons that could establish functional differences between these two proteins. To address this challenge, we have developed a single-molecule colocalization binding titration assay (smCoBRA) that can quantitatively characterize full-length otoferlin from mammalian cell lysate. Using smCoBRA, we found that, although both otoferlin and synaptotagmin bind membrane fusion SNARE proteins, only otoferlin interacts with the L-type calcium channel Cav1.3, showing a significant difference between the synaptic proteins. Furthermore, otoferlin was found capable of interacting with multiple SNARE and Cav1.3 proteins simultaneously, forming a heterooligomer complex. We also found that a deafness-causing missense mutation in otoferlin attenuates binding between otoferlin and Cav1.3, suggesting that deficiencies in this interaction may form the basis for otoferlin-related hearing loss. Based on our results, we propose a model in which otoferlin acts as a calcium-sensitive scaffolding protein, localizing SNARE proteins proximal to the calcium channel so as to synchronize calcium influx with membrane fusion. Our findings also provide a molecular-level explanation for the observation that synaptotagmin and otoferlin are not functionally redundant. This study also validates a generally applicable methodology for quantitatively characterizing large, multivalent membrane proteins.

Dysferlin Binds SNAREs (Soluble N-Ethylmaleimide-sensitive Factor (NSF) Attachment Protein Receptors) and Stimulates Membrane Fusion in a Calcium-sensitive Manner.

Resealing of tears in the sarcolemma of myofibers is a necessary step in the repair of muscle tissue. Recent work suggests a critical role for dysferlin in the membrane repair process and that mutations in dysferlin are responsible for limb girdle muscular dystrophy 2B and Miyoshi myopathy. Beyond membrane repair, dysferlin has been linked to SNARE-mediated exocytotic events including cytokine release and acid sphingomyelinase secretion. However, it is unclear whether dysferlin regulates SNARE-mediated membrane fusion. In this study we demonstrate a direct interaction between dysferlin and the SNARE proteins syntaxin 4 and SNAP-23. In addition, analysis of FRET and in vitro reconstituted lipid mixing assays indicate that dysferlin accelerates syntaxin 4/SNAP-23 heterodimer formation and SNARE-mediated lipid mixing in a calcium-sensitive manner. These results support a function for dysferlin as a calcium-sensing SNARE effector for membrane fusion events.

Otoferlin deficiency in zebrafish results in defects in balance and hearing: rescue of the balance and hearing phenotype with full-length and truncated forms of mouse otoferlin.

Sensory hair cells convert mechanical motion into chemical signals. Otoferlin, a six-C2 domain transmembrane protein linked to deafness in humans, is hypothesized to play a role in exocytosis at hair cell ribbon synapses. To date, however, otoferlin has been studied almost exclusively in mouse models, and no rescue experiments have been reported. Here we describe the phenotype associated with morpholino-induced otoferlin knockdown in zebrafish and report the results of rescue experiments conducted with full-length and truncated forms of otoferlin. We found that expression of otoferlin occurs early in development and is restricted to hair cells and the midbrain. Immunofluorescence microscopy revealed localization to both apical and basolateral regions of hair cells. Knockdown of otoferlin resulted in hearing and balance defects, as well as locomotion deficiencies. Further, otoferlin morphants had uninflated swim bladders. Rescue experiments conducted with mouse otoferlin restored hearing, balance, and inflation of the swim bladder. Remarkably, truncated forms of otoferlin retaining the C-terminal C2F domain also rescued the otoferlin knockdown phenotype, while the individual N-terminal C2A domain did not. We conclude that otoferlin plays an evolutionarily conserved role in vertebrate hearing and that truncated forms of otoferlin can rescue hearing and balance.

Characterization of the lipid binding properties of Otoferlin reveals specific interactions between PI(4,5)P2 and the C2C and C2F domains.

Otoferlin is a transmembrane protein consisting of six C2 domains, proposed to act as a calcium sensor for exocytosis. Although otoferlin is believed to bind calcium and lipids, the lipid specificity and identity of the calcium binding domains are controversial. Further, it is currently unclear whether the calcium binding affinity of otoferlin quantitatively matches the maximal intracellular presynaptic calcium concentrations of ∼30-50 µM known to elicit exocytosis. To characterize the calcium and lipid binding properties of otoferlin, we used isothermal titration calorimetry (ITC), liposome sedimentation assays, and fluorescence spectroscopy. Analysis of ITC data indicates that with the exception of the C2A domain, the C2 domains of otoferlin bind multiple calcium ions with moderate (Kd = 25-95 µM) and low affinities (Kd = 400-700 µM) in solution. However, in the presence of liposomes, the calcium sensitivity of the domains increased by up to 10-fold. It was also determined that calcium enhanced liposome binding for domains C2B-C2E, whereas the C2F domain bound liposomes in a calcium-independent manner. Mutations that abrogate calcium binding in C2F do not disrupt liposome binding, supporting the conclusion that the interaction of the C2F domain with phosphatidylserine is calcium-independent. Further, domains C2C and C2F, not domains C2A, C2B, C2D, and C2E, bound phosphatidylinositol 4,5-bisphosphate 1,2-dioleoyl-sn-glycero-3-phospho(1'-myoinositol-4',5'-bisphosphate) [PI(4,5)P2], which preferentially steered them toward liposomes harboring PI(4,5)P2. Remarkably, lysine mutations L478A and L480A in C2C selectively weaken the PI(4,5)P2 interaction while leaving phosphatidylserine binding unaffected. Finally, shifts in the emission spectra of an environmentally sensitive fluorescent unnatural amino acid indicate that the calcium binding loops of the C2F domain directly interact with the lipid bilayer of negatively charged liposomes in a calcium-independent manner. On the basis of these results, we propose that the C2F and C2C domains of otoferlin preferentially bind PI(4,5)P2 and that PI(4,5)P2 may serve to target otoferlin to the presynapse in a calcium-independent manner. This positioning would facilitate fast calcium-dependent exocytosis at the hair cell synapse.

Quantitation of the calcium and membrane binding properties of the C2 domains of dysferlin.

Dysferlin is a large membrane protein involved in calcium-triggered resealing of the sarcolemma after injury. Although it is generally accepted that dysferlin is Ca(2+) sensitive, the Ca(2+) binding properties of dysferlin have not been characterized. In this study, we report an analysis of the Ca(2+) and membrane binding properties of all seven C2 domains of dysferlin as well as a multi-C2 domain construct. Isothermal titration calorimetry measurements indicate that all seven dysferlin C2 domains interact with Ca(2+) with a wide range of binding affinities. The C2A and C2C domains were determined to be the most sensitive, with Kd values in the tens of micromolar, whereas the C2D domain was least sensitive, with a near millimolar Kd value. Mutagenesis of C2A demonstrates the requirement for negatively charged residues in the loop regions for divalent ion binding. Furthermore, dysferlin displayed significantly lower binding affinity for the divalent cations magnesium and strontium. Measurement of a multidomain construct indicates that the solution binding affinity does not change when C2 domains are linked. Finally, sedimentation assays suggest all seven C2 domains bind lipid membranes, and that Ca(2+) enhances but is not required for interaction. This report reveals for the first time, to our knowledge, that all dysferlin domains bind Ca(2+) albeit with varying affinity and stoichiometry.

The C2 domains of otoferlin, dysferlin, and myoferlin alter the packing of lipid bilayers.

Ferlins are large multi-C2 domain membrane proteins involved in membrane fusion and fission events. In this study, we investigate the effects of binding of the C2 domains of otoferlin, dysferlin, and myoferlin on the structure of lipid bilayers. Fluorescence measurements indicate that multi-C2 domain constructs of myoferlin, dysferlin, and otoferlin change the lipid packing of both small unilamellar vesicles and giant plasma membrane vesicles. The activities of these proteins were enhanced in the presence of calcium and required negatively charged lipids like phosphatidylserine or phosphatidylglycerol for activity. Experiments with individual domains uncovered functional differences between the C2A domain of otoferlin and those of dysferlin and myoferlin, and truncation studies suggest that the effects of each subsequent C2 domain on lipid ordering appear to be additive. Finally, we demonstrate that the activities of these proteins on membranes are insensitive to high salt concentrations, suggesting a nonelectrostatic component to the interaction between ferlin C2 domains and lipid bilayers. Together, the data indicate that dysferlin, otoferlin, and myoferlin do not merely passively adsorb to membranes but actively sculpt lipid bilayers, which would result in highly curved or distorted membrane regions that could facilitate membrane fusion, membrane fission, or recruitment of other membrane-trafficking proteins.

Mechanism and function of synaptotagmin-mediated membrane apposition.

Synaptotagmin-1 is a Ca(2+) sensor that triggers synchronous neurotransmitter release. The first documented biochemical property of synaptotagmin-1 was its ability to aggregate membranes in response to Ca(2+). However, the mechanism and function of this process were poorly understood. Here we show that synaptotagmin-1-mediated vesicle aggregation is driven by trans interactions between synaptotagmin-1 molecules bound to different membranes. We found a strong correlation between the ability of Ca(2+)-bound synaptotagmin-1 to aggregate vesicles and to stimulate SNARE-mediated membrane fusion. Moreover, artificial aggregation of membranes-using non-synaptotagmin proteins-also efficiently promoted fusion of SNARE-bearing liposomes. Finally, using a modified fusion assay, we observed that synaptotagmin-1 drove the assembly of otherwise non-fusogenic individual t-SNARE proteins into fusion-competent heterodimers, independently of aggregation. Thus, membrane aggregation and t-SNARE assembly appear to be two key aspects of fusion reactions that are regulated by Ca(2+)-bound synaptotagmin-1 and catalyzed by SNAREs.

Otoferlin is a calcium sensor that directly regulates SNARE-mediated membrane fusion.

Otoferlin is a large multi-C2 domain protein proposed to act as a calcium sensor that regulates synaptic vesicle exocytosis in cochlear hair cells. Although mutations in otoferlin have been associated with deafness, its contribution to neurotransmitter release is unresolved. Using recombinant proteins, we demonstrate that five of the six C2 domains of otoferlin sense calcium with apparent dissociation constants that ranged from 13-25 µM; in the presence of membranes, these apparent affinities increase by up to sevenfold. Using a reconstituted membrane fusion assay, we found that five of the six C2 domains of otoferlin stimulate membrane fusion in a calcium-dependent manner. We also demonstrate that a calcium binding-deficient form of the C2C domain is incapable of stimulating membrane fusion, further underscoring the importance of calcium for the protein's function. These results demonstrate for the first time that otoferlin is a calcium sensor that can directly regulate soluble N-ethyl-maleimide sensitive fusion protein attachment protein receptor-mediated membrane fusion reactions.

Synaptotagmin-mediated bending of the target membrane is a critical step in Ca(2+)-regulated fusion.

Decades ago it was proposed that exocytosis involves invagination of the target membrane, resulting in a highly localized site of contact between the bilayers destined to fuse. The vesicle protein synaptotagmin-I (syt) bends membranes in response to Ca(2+), but whether this drives localized invagination of the target membrane to accelerate fusion has not been determined. Previous studies relied on reconstituted vesicles that were already highly curved and used mutations in syt that were not selective for membrane-bending activity. Here, we directly address this question by utilizing vesicles with different degrees of curvature. A tubulation-defective syt mutant was able to promote fusion between highly curved SNARE-bearing liposomes but exhibited a marked loss of activity when the membranes were relatively flat. Moreover, bending of flat membranes by adding an N-BAR domain rescued the function of the tubulation-deficient syt mutant. Hence, syt-mediated membrane bending is a critical step in membrane fusion.

Embryonic cardiomyocytes beat best on a matrix with heart-like elasticity: scar-like rigidity inhibits beating.

Fibrotic rigidification following a myocardial infarct is known to impair cardiac output, and it is also known that cardiomyocytes on rigid culture substrates show a progressive loss of rhythmic beating. Here, isolated embryonic cardiomyocytes cultured on a series of flexible substrates show that matrices that mimic the elasticity of the developing myocardial microenvironment are optimal for transmitting contractile work to the matrix and for promoting actomyosin striation and 1-Hz beating. On hard matrices that mechanically mimic a post-infarct fibrotic scar, cells overstrain themselves, lack striated myofibrils and stop beating; on very soft matrices, cells preserve contractile beating for days in culture but do very little work. Optimal matrix leads to a strain match between cell and matrix, and suggests dynamic differences in intracellular protein structures. A 'cysteine shotgun' method of labeling the in situ proteome reveals differences in assembly or conformation of several abundant cytoskeletal proteins, including vimentin, filamin and myosin. Combined with recent results, which show that stem cell differentiation is also highly sensitive to matrix elasticity, the methods and analyses might be useful in the culture and assessment of cardiogenesis of both embryonic stem cells and induced pluripotent stem cells. The results described here also highlight the need for greater attention to fibrosis and mechanical microenvironments in cell therapy and development.

Forced unfolding of proteins within cells.

To identify cytoskeletal proteins that change conformation or assembly within stressed cells, in situ labeling of sterically shielded cysteines with fluorophores was analyzed by fluorescence imaging, quantitative mass spectrometry, and sequential two-dye labeling. Within red blood cells, shotgun labeling showed that shielded cysteines in the two isoforms of the cytoskeletal protein spectrin were increasingly labeled as a function of shear stress and time, indicative of forced unfolding of specific domains. Within mesenchymal stem cells-as a prototypical adherent cell-nonmuscle myosin IIA and vimentin are just two of the cytoskeletal proteins identified that show differential labeling in tensed versus drug-relaxed cells. Cysteine labeling of proteins within live cells can thus be used to fluorescently map out sites of molecular-scale deformation, and the results also suggest means to colocalize signaling events such as phosphorylation with forced unfolding.