Robin Wray

Activity-Driven Dendritic Remodeling Requires Microtubule-Associated Protein 1A

Activity-prompted dendritic remodeling leads to calcium-influx-dependent activation of signaling pathways within minutes and gene transcription within hours. However, dendrite growth continues for days and requires extension and stabilization of the cytoskeleton in nascent processes. In addition to binding microtubules, microtubule-associated proteins (MAPs) associate with the actin cytoskeleton, anchor ion channels and signaling complexes, and modulate synaptic growth. MAP2 is predominantly dendritic. MAP1B is at postsynaptic densities (PSD) and modulates ion channel activity, in addition to affecting axon growth. Less is known about MAP1A, but it is also enriched in dendrites at input locations, including PSDs where MAP1A associates with channel complexes and the calcium sensor caldendrin. MAP1A rescued hearing loss in tubby mice. Here we show that MAP1A becomes enriched in dendrites concurrently with dendritic branching and synapse formation in the developing brain; that synaptic activity is required for establishing mature MAP1A expression levels; and that MAP1A expression is required for activity-dependent growth, branching, and stabilization of the dendritic arbor.

S. aureus MscL Is a Pentamer In Vivo but of Variable Stoichiometries In Vitro: Implications for Detergent- Solubilized Membrane Proteins

Detergent-induced rearrangements of membrane-protein subunits explain why two MscL channel stoichiometries have been resolved by X-ray crystallography - but S. aureus MscL is truly a pentamer in vivo.

On the Structure of the N-Terminal Domain of the MscL Channel: Helical Bundle or Membrane Interface

The mechanosensitive channel of large conductance, MscL, serves as a biological emergency release valve protecting bacteria from acute osmotic downshock and is to date the best characterized mechanosensitive channel. A well-recognized and supported model for Escherichia coli MscL gating proposes that the N-terminal 11 amino acids of this protein form a bundle of amphipathic helices in the closed state that functionally serves as a cytoplasmic second gate. However, a recently reexamined crystal structure of a closed state of the Mycobacterium tuberculosis MscL shows these helices running along the cytoplasmic surface of the membrane. Thus, it is unclear if one structural model is correct or if they both reflect valid closed states. Here, we have systematically reevaluated this region utilizing cysteine-scanning, in vivo functional characterization, in vivo SCAM, electrophysiological studies, and disulfide-trapping experiments. The disulfide-trapping pattern and functional studies do not support the helical bundle and second-gate hypothesis but correlate well with the proposed structure for M. tuberculosis MscL. We propose a functional model that is consistent with the collective data.

Intragenic suppression of gain‐of‐function mutations in the Escherichia coli mechanosensitive channel, MscL

Mechanosensitive channels play an important role in protecting bacterial cells from osmotic downshock by serving as biological ‘pressure release valves’. One of these channels, MscL, is found throughout the bacterial kingdom, but has been most studied in Escherichia coli. The E. coli MscL is a 136‐amino‐acid protein organized as a homopentamer with each subunit containing two transmembrane segments. Previous studies have shown that several residues, including V23 and G26, are essential for normal function of MscL; very severe gain‐of‐function phenotypes in which cell growth slows or is arrested can result from residue substitutions at these positions. Through random mutagenesis and growth selection, we have generated intragenic suppressors of the V23A and G26S mutations. The suppressor mutants have been characterized by growth phenotype, Western blot and patch clamp. Most of the mutations that render phenotypic suppression are located in the transmembrane domains with additional sites lying in the periplasmic loop. In contrast, only one mutation is found in the amino‐terminal S1 domain, and none is found within the carboxyl‐terminal domain. Not only have these findings revealed functional domains and subdomains critical for MscL function, but they also predict a pair of residues that interact directly during channel opening.

The oligomeric state of the truncated mechanosensitive channel of large conductance shows no variance in vivo

The mechanosensitive channel of large conductance (MscL) from E. coli serves as an emergency release valve allowing the cell to survive acute osmotic downshock. It is one of the best studied mechanosensitive channels and serves as a paradigm for how a protein can sense and respond to membrane tension. Two MscL crystal structures of the orthologs M. tuberculosis and S. aureus have been solved showing pentameric and tetrameric structures, respectively. Several studies followed to understand whether the discrepancy in their stoichiometry was a species difference or a consequence of the protein manipulation for crystallization. Two independent studies now agree that the full‐length S. aureus MscL is actually a pentamer, not tetramer. While detergents appear to play a role in modifying the oligomeric state of the protein, a cytoplasmic helical bundle has also been implicated. Here, we evaluate the role of the C‐terminal region of S. aureus MscL in the oligomerization of the channel in native membranes by using an in vivo disulfide‐trapping technique. We find that the oligomeric state of S. aureus MscLs with different C‐terminal truncations, including the one used to obtain the tetrameric S. aureus MscL crystal structure, are pentamers in vivo. Thus, the C‐terminal domain of the S. aureus protein only plays a critical role in the oligomeric state of the SaMscL protein when it is solubilized in detergent.

An open-pore structure of the mechanosensitive channel MscL derived by determining transmembrane domain interactions upon gating.

Mechanosensation, the ability to detect mechanical forces, underlies the senses of hearing, balance, touch, and pain, as well as renal and cardiovascular regulation. Although the sensors are thought to be channels, relatively little is known about eukaryotic mechanosensitive channels or their molecular mechanisms. Thus, because of its tractable nature, a bacterial mechanosensitive channel that serves as an in vivo osmotic “emergency release valve,” MscL, has become a paradigm of how a mechanosensitive channel can sense and respond to membrane tension. Here, we have determined the structural rearrangements and interactions between transmembrane domains of MscL that occur upon gating. We utilize an electrostatic repulsion test: If two residues approach upon gating we predicted that substituting like‐charges at those sites would inhibit gating. The in vivo growth and viability and in vitro vesicular flux and electrophysiological data all support the hypothesis that residues G26 and I92 directly interact upon gating. The resulting model predicted other interacting residues. One of these sets, V23 and I96, was confirmed to truly interact upon gating by disulfide trapping as well as the electrostatic repulsion test. Together, the data strongly suggest a model for structural transitions and residue‐residue proximities that occur upon MscL gating.—Li, Y.,Wray, R., Eaton, C., Blount, P. An open‐pore structure of the mechano‐sensitive channel MscL derived by determining transmembrane domain interactions upon gating. FASEB J. 23, 2197–2204 (2009)

Three routes to modulate the pore size of the Mscl channel/nanovalve

MscL is a bacterial mechanosensitive channel that protects cells from lysis upon acute decrease in external osmotic environment. It is one of the best characterized mechanosensors known, thus serving as a paradigm of how such molecules sense and respond to stimuli. In addition, the fact that it can be genetically modified, expressed, isolated, and manipulated has led to its proposed use as a triggered nanovalve for various functions including sensors within microelectronic array chips, as well as vesicular-based targeted drug release. X-ray crystallography reveals a homopentameric complex with each subunit containing two transmembrane α-helices (TM1 and TM2) and a single carboxyl terminal α-helix arranging within the complex to form a 5-fold cytoplasmic bundle (CB), whose function and stability remain unclear. In this study, we show three routes that throttle the open channel conductance. When the linker between the TM2 and CB domain is shortened by deletions or constrained by either cross-linking or heavy metal coordination, the conductance of the channel is reduced; in the later two cases, even reversibly. While they have implications for the stability of the CB, these data also provide routes for engineering MscL sensors that are more versatile for potential nanotech devices.

Streptomycin potency is dependent on MscL channel expression

The antibiotic streptomycin is widely used in the treatment of microbial infections. The primary mechanism of action is inhibition of translation by binding to the ribosome, but how it enters the bacterial cell is unclear. Early in the study of this antibiotic, a mysterious streptomycin-induced K+-efflux preceding any decrease in viability was observed; it was speculated that this changed the electrochemical gradient such that streptomycin better accessed the cytoplasm. Here we use a high throughput screen to search for compounds targeting the mechanosensitive channel of large conductance (MscL) and find dihydrostreptomycin among the “hits”. Furthermore, we find that MscL is not only necessary for the previously described streptomycin-induced K+-efflux, but also directly increases MscL activity in electrophysiological studies. The data suggest that gating MscL is a novel mode of action of dihydrostreptomycin, and that MscL’s large pore may provide a mechanism for cell entry.

Improving the Design of a MscL-Based Triggered Nanovalve

The mechanosensitive channel of large conductance, MscL, has been proposed as a triggered nanovalve to be used in drug release and other nanodevices. It is a small homopentameric bacterial protein that has the largest gated pore known: greater than 30 Å. Large molecules, even small proteins can be released through MscL. Although MscL normally gates in response to membrane tension, early studies found that hydrophilic or charged residue substitutions near the constriction of the channel leads to pore opening. Researchers have successfully changed the modality of MscL to open to stimuli such as light by chemically modifying a single residue, G22, within the MscL pore. Here, by utilizing in vivo, liposome efflux, and patch clamp assays we compared modification of G22 with that of another neighboring residue, G26, and demonstrate that modifying G26 may be a better choice for triggered nanovalves used for triggered vesicular release of compounds.

An in vivo screen reveals protein-lipid interactions crucial for gating a mechanosensitive channel

The bacterial mechanosensitive channel MscL is the best‐studied mechanosensor, thus serving as a paradigm of how a protein senses and responds to mechanical force. Models for the transition of Escherichia coli MscL from closed to open states propose a tilting of the transmembrane domains in the plane of the membrane, suggesting dynamic protein‐lipid interactions. Here, we used a rapid in vivo assay to assess the function of channels that were post‐translationally modified at several different sites in a region just distal to the cytoplasmic end of the second transmembrane helix. We utilized multiple probes with various affinities for the membrane environment. The in vivo functional data, combined with site‐directed mutagenesis, single‐channel analyses, and tryptophan fluorescence measurements, confirmed that lipid interactions within this region are critical for MscL gating. The data suggest a model in which this region acts as an anchor for the transmembrane domain tilting during gating. Furthermore, the conservation of analogous motifs among many other channels suggests a conserved pro‐tein‐lipid dynamic mechanism.—Iscla, I., Wray, R., Blount, P. An in vivo screen reveals protein‐lipid interactions crucial for gating a mechanosensitive channel. FASEB J. 25, 694–702 (2011). www.fasebj.org