Steven S. Vogel

Defective membrane repair in dysferlin-deficient muscular dystrophy

Muscular dystrophy includes a diverse group of inherited muscle diseases characterized by wasting and weakness of skeletal muscle. Mutations in dysferlin are linked to two clinically distinct muscle diseases, limb-girdle muscular dystrophy type 2B and Miyoshi myopathy, but the mechanism that leads to muscle degeneration is unknown. Dysferlin is a homologue of the Caenorhabditis elegans fer-1 gene, which mediates vesicle fusion to the plasma membrane in spermatids. Here we show that dysferlin-null mice maintain a functional dystrophin–glycoprotein complex but nevertheless develop a progressive muscular dystrophy. In normal muscle, membrane patches enriched in dysferlin can be detected in response to sarcolemma injuries. In contrast, there are sub-sarcolemmal accumulations of vesicles in dysferlin-null muscle. Membrane repair assays with a two-photon laser-scanning microscope demonstrated that wild-type muscle fibres efficiently reseal their sarcolemma in the presence of Ca2+. Interestingly, dysferlin-deficient muscle fibres are defective in Ca2+-dependent sarcolemma resealing. Membrane repair is therefore an active process in skeletal muscle fibres, and dysferlin has an essential role in this process. Our findings show that disruption of the muscle membrane repair machinery is responsible for dysferlin-deficient muscle degeneration, and highlight the importance of this basic cellular mechanism of membrane resealing in human disease.

Lysolipids reversibly inhibit Ca2+‐, GTP‐ and pH‐dependent fusion of biological membranes

Membrane fusion in exocytosis, intracellular trafficking, and enveloped viral infection is thought to be mediated by specialized proteins acting to merge membrane lipid bilayers. We now show that one class of naturally‐occurring phospholipids, lysolipids, inhibits fusion between cell membranes, organelles, and between organelles and plasma membrane. Inhibition was reversible, did not correlate with lysis, and could be attributed to the molecular shape of lysolipids rather than to any specific chemical moiety. Fusion was arrested at a stage preceding fusion pore formation. Our results are consistent with the hypothesis that biological fusion, irrespective of trigger, involves the formation of a highly bent intermediate between membranes, the fusion stalk.

Analysis of pancreatic development in living transgenic zebrafish embryos.

Using DNA constructs containing regulatory sequences of the zebrafish Pdx-1 and insulin genes, germline transgenic zebrafish expressing the green fluorescent protein (GFP) reporter gene in the pancreas were generated. For both constructs, the GFP expression patterns in transgenic embryos were consistent with the mRNA expression patterns detected by RNA in situ hybridization. A deletion promoter analysis revealed that positive and negative cis-acting elements were involved in regulation of insulin gene expression. Three-dimensional reconstructions imaged from living embryos using two-photon laser-scanning microscopy (TPLSM) demonstrated that the zebrafish pancreas is formed from a single dorsal pancreatic cell mass. This is in contrast to mammals where the pancreas derives from both dorsal and ventral anlage. These transgenic fish should be useful for in vivo studies of factors involved in specifying and regulating pancreatic development and function.

The endomembrane requirement for cell surface repair

The capacity to reseal a plasma membrane disruption rapidly is required for cell survival in many physiological environments. Intracellular membrane (endomembrane) is thought to play a central role in the rapid resealing response. We here directly compare the resealing response of a cell that lacks endomembrane, the red blood cell, with that of several nucleated cells possessing an abundant endomembrane compartment. RBC membrane disruptions inflicted by a mode-locked Ti:sapphire laser, even those initially smaller than hemoglobin, failed to reseal rapidly. By contrast, much larger laser-induced disruptions made in sea urchin eggs, fibroblasts, and neurons exhibited rapid, Ca2+-dependent resealing. We conclude that rapid resealing is not mediated by simple physiochemical mechanisms; endomembrane is required.

Dysferlin stabilizes stress-induced Ca2+ signaling in the transverse tubule membrane

Significance Muscular dystrophies linked to the genetic absence or mutations of dysferlin are currently without a relevant therapy. Dysferlin is thought to mediate membrane repair in skeletal muscle, but its localization and specific functions remain controversial. Here we show that dysferlin is enriched in the transverse tubule membrane of skeletal muscle and demonstrate that, in its absence, mechanical stress leads to calcium-dependent muscle injury. Furthermore, we demonstrate that treatment of dysferlin-deficient muscle with the calcium channel blocker diltiazem reduces in vitro experimental and in vivo contraction-induced muscle damage. As diltiazem is approved for clinical use, our results suggest a potential new therapeutic avenue for patients diagnosed with dysferlinopathies. Dysferlinopathies, most commonly limb girdle muscular dystrophy 2B and Miyoshi myopathy, are degenerative myopathies caused by mutations in the DYSF gene encoding the protein dysferlin. Studies of dysferlin have focused on its role in the repair of the sarcolemma of skeletal muscle, but dysferlin’s association with calcium (Ca2+) signaling proteins in the transverse (t-) tubules suggests additional roles. Here, we reveal that dysferlin is enriched in the t-tubule membrane of mature skeletal muscle fibers. Following experimental membrane stress in vitro, dysferlin-deficient muscle fibers undergo extensive functional and structural disruption of the t-tubules that is ameliorated by reducing external [Ca2+] or blocking L-type Ca2+ channels with diltiazem. Furthermore, we demonstrate that diltiazem treatment of dysferlin-deficient mice significantly reduces eccentric contraction-induced t-tubule damage, inflammation, and necrosis, which resulted in a concomitant increase in postinjury functional recovery. Our discovery of dysferlin as a t-tubule protein that stabilizes stress-induced Ca2+ signaling offers a therapeutic avenue for limb girdle muscular dystrophy 2B and Miyoshi myopathy patients.

Anomalous Surplus Energy Transfer Observed with Multiple FRET Acceptors

Background Förster resonance energy transfer (FRET) is a mechanism where energy is transferred from an excited donor fluorophore to adjacent chromophores via non-radiative dipole-dipole interactions. FRET theory primarily considers the interactions of a single donor-acceptor pair. Unfortunately, it is rarely known if only a single acceptor is present in a molecular complex. Thus, the use of FRET as a tool for measuring protein-protein interactions inside living cells requires an understanding of how FRET changes with multiple acceptors. When multiple FRET acceptors are present it is assumed that a quantum of energy is either released from the donor, or transferred in toto to only one of the acceptors present. The rate of energy transfer between the donor and a specific acceptor (kD→A) can be measured in the absence of other acceptors, and these individual FRET transfer rates can be used to predict the ensemble FRET efficiency using a simple kinetic model where the sum of all FRET transfer rates is divided by the sum of all radiative and non-radiative transfer rates. Methodology/Principal Findings The generality of this approach was tested by measuring the ensemble FRET efficiency in two constructs, each containing a single fluorescent-protein donor (Cerulean) and either two or three FRET acceptors (Venus). FRET transfer rates between individual donor-acceptor pairs within these constructs were calculated from FRET efficiencies measured after systematically introducing point mutations to eliminate all other acceptors. We find that the amount of energy transfer observed in constructs having multiple acceptors is significantly greater than the FRET efficiency predicted from the sum of the individual donor to acceptor transfer rates. Conclusions/Significance We conclude that either an additional energy transfer pathway exists when multiple acceptors are present, or that a theoretical assumption on which the kinetic model prediction is based is incorrect.

Energy migration alters the fluorescence lifetime of Cerulean: implications for fluorescence lifetime imaging Forster resonance energy transfer measurements

Forster resonance energy transfer (FRET) is a physical phenomenon used to study molecular interactions in living cells. Changes in the fluorescence lifetime of proteins genetically tagged with a donor fluorophore, such as cyan fluorescent protein or Cerulean, are used to measure energy transfer to a protein tagged with an acceptor fluorophore (yellow fluorescent protein or Venus). Increased transfer efficiency is usually interpreted as closer proximity. Resonance energy transfer is also possible between identical fluorophores. This form of FRET is called energy migration resonance energy transfer (EM-RET). Theoretically, EM-RET should not alter the lifetime or emission spectrum measured from a population of fluorophores. We find a change in the fluorescent lifetime of Cerulean that correlates with energy migration and can result in significant errors when using Cerulean as a donor to measure FRET efficiencies based on fluorescence lifetimes [corrected]

Fluorescence Polarization and Fluctuation Analysis Monitors Subunit Proximity, Stoichiometry, and Protein Complex Hydrodynamics

Förster resonance energy transfer (FRET) microscopy is frequently used to study protein interactions and conformational changes in living cells. The utility of FRET is limited by false positive and negative signals. To overcome these limitations we have developed Fluorescence Polarization and Fluctuation Analysis (FPFA), a hybrid single-molecule based method combining time-resolved fluorescence anisotropy (homo-FRET) and fluorescence correlation spectroscopy. Using FPFA, homo-FRET (a 1-10 nm proximity gauge), brightness (a measure of the number of fluorescent subunits in a complex), and correlation time (an attribute sensitive to the mass and shape of a protein complex) can be simultaneously measured. These measurements together rigorously constrain the interpretation of FRET signals. Venus based control-constructs were used to validate FPFA. The utility of FPFA was demonstrated by measuring in living cells the number of subunits in the α-isoform of Venus-tagged calcium-calmodulin dependent protein kinase-II (CaMKIIα) holoenzyme. Brightness analysis revealed that the holoenzyme has, on average, 11.9 ± 1.2 subunit, but values ranged from 10-14 in individual cells. Homo-FRET analysis simultaneously detected that catalytic domains were arranged as dimers in the dodecameric holoenzyme, and this paired organization was confirmed by quantitative hetero-FRET analysis. In freshly prepared cell homogenates FPFA detected only 10.2 ± 1.3 subunits in the holoenzyme with values ranging from 9-12. Despite the reduction in subunit number, catalytic domains were still arranged as pairs in homogenates. Thus, FPFA suggests that while the absolute number of subunits in an auto-inhibited holoenzyme might vary from cell to cell, the organization of catalytic domains into pairs is preserved.

The Impact of Heterogeneity and Dark Acceptor States on FRET: Implications for Using Fluorescent Protein Donors and Acceptors

Förster resonance energy transfer (FRET) microscopy is widely used to study protein interactions in living cells. Typically, spectral variants of the Green Fluorescent Protein (FPs) are incorporated into proteins expressed in cells, and FRET between donor and acceptor FPs is assayed. As appreciable FRET occurs only when donors and acceptors are within 10 nm of each other, the presence of FRET can be indicative of aggregation that may denote association of interacting species. By monitoring the excited-state (fluorescence) decay of the donor in the presence and absence of acceptors, dual-component decay analysis has been used to reveal the fraction of donors that are FRET positive (i.e., in aggregates)._However, control experiments using constructs containing both a donor and an acceptor FP on the same protein repeatedly indicate that a large fraction of these donors are FRET negative, thus rendering the interpretation of dual-component analysis for aggregates between separately donor-containing and acceptor-containing proteins problematic. Using Monte-Carlo simulations and analytical expressions, two possible sources for such anomalous behavior are explored: 1) conformational heterogeneity of the proteins, such that variations in the distance separating donor and acceptor FPs and/or their relative orientations persist on time-scales long in comparison with the excited-state lifetime, and 2) FP dark states.

Quantitative linear unmixing of CFP and YFP from spectral images acquired with two‐photon excitation

Spectrally distinct fluorescent proteins (FPs) have been developed permitting the visualization of several proteins simultaneously in living cells. The emission spectra of FPs, in most cases, overlap, making signal separation based on filter technology inefficient and in cases of bleed‐through, inaccurate. Spectral imaging can overcome these obstacles through a process called linear unmixing. Given a complex spectra composed of multiple fluorophores, linear unmixing can reduce the complex signal to its individual, weighted, component spectra. Spectral imaging with two‐photon excitation allows the collection of nontruncated emission spectra. The accuracy of linear unmixing under these conditions needs to be evaluated.