Paul R. Rohde

Regulation of Cholesterol Homeostasis and Lipid Metabolism in Skeletal Muscle by Liver X Receptors

Recent studies have identified the liver X receptors (LXRα and LXRβ) as important regulators of cholesterol and lipid metabolism. Although originally identified as liver-enriched transcription factors, LXRs are also expressed in skeletal muscle, a tissue that accounts for ∼40% of human total body weight and is the major site of glucose utilization and fatty acid oxidation. Nevertheless, no studies have yet addressed the functional role of LXRs in muscle. In this work we utilize a combination of in vivoand in vitro analysis to demonstrate that LXRs can functionally regulate genes involved in cholesterol metabolism in skeletal muscle. Furthermore we show that treatment of muscle cellsin vitro with synthetic agonists of LXR increases the efflux of intracellular cholesterol to extracellular acceptors such as high density lipoprotein, thus identifying this tissue as a potential important regulator of reverse cholesterol transport and high density lipoprotein levels. Additionally we demonstrate that LXRα and a subset of LXR target genes are induced during myogenesis, suggesting a role for LXR-dependent signaling in the differentiation process.

Removal of the mechanoprotective influence of the cytoskeleton reveals PIEZO1 is gated by bilayer tension

Mechanosensitive ion channels are force-transducing enzymes that couple mechanical stimuli to ion flux. Understanding the gating mechanism of mechanosensitive channels is challenging because the stimulus seen by the channel reflects forces shared between the membrane, cytoskeleton and extracellular matrix. Here we examine whether the mechanosensitive channel PIEZO1 is activated by force-transmission through the bilayer. To achieve this, we generate HEK293 cell membrane blebs largely free of cytoskeleton. Using the bacterial channel MscL, we calibrate the bilayer tension demonstrating that activation of MscL in blebs is identical to that in reconstituted bilayers. Utilizing a novel PIEZO1–GFP fusion, we then show PIEZO1 is activated by bilayer tension in bleb membranes, gating at lower pressures indicative of removal of the cortical cytoskeleton and the mechanoprotection it provides. Thus, PIEZO1 channels must sense force directly transmitted through the bilayer.

Bilayer-Mediated Clustering and Functional Interaction of MscL Channels

Mechanosensitive channels allow bacteria to respond to osmotic stress by opening a nanometer-sized pore in the cellular membrane. Although the underlying mechanism has been thoroughly studied on the basis of individual channels, the behavior of channel ensembles has yet to be elucidated. This work reveals that mechanosensitive channels of large conductance (MscL) exhibit a tendency to spatially cluster, and demonstrates the functional relevance of clustering. We evaluated the spatial distribution of channels in a lipid bilayer using patch-clamp electrophysiology, fluorescence and atomic force microscopy, and neutron scattering and reflection techniques, coupled with mathematical modeling of the mechanics of a membrane crowded with proteins. The results indicate that MscL forms clusters under a wide range of conditions. MscL is closely packed within each cluster but is still active and mechanosensitive. However, the channel activity is modulated by the presence of neighboring proteins, indicating membrane-mediated protein-protein interactions. Collectively, these results suggest that MscL self-assembly into channel clusters plays an osmoregulatory functional role in the membrane.

MOLECULAR DETECTION OF PROSTATE CELLS IN EJACULATE AND URETHRAL WASHINGS IN MEN WITH SUSPECTED PROSTATE CANCER

PURPOSE To determine whether prostatic cells were normally present in ejaculate and if the sensitivity and specificity of the detection of malignant prostate cells in ejaculate and urethral washings from men with suspected prostate cancer could be improved using the more sensitive molecular technique of reverse transcriptase-polymerase chain reaction (RT-PCR). MATERIALS AND METHODS RT-PCR for prostate-specific antigen (PSA), prostate-specific membrane antigen (PSM) and Apoliprotein D (3 putative prostate-specific and/or cancer-specific markers) was performed on RNA extracts of ejaculate (80) and urethral washings (52) from 77 men with suspected prostate cancer and 12 young controls (<30 years of age) and urines from 5 men who had radical prostatectomies and 10 women. RESULTS PSA, PSM and Apolipoprotein D expression was detected in ejaculates and urethral washings from both patient and control groups. No differences were observed in the results obtained for 58 men with suspected or 19 men with confirmed prostate cancer or the 18 vasectomized men within the patient group. Urines from the 5 men who had radical prostatectomies and 10 women were all negative for PSA, but PSM was detected in 2 female urines and in 3 radical prostatectomy samples. As few as 10 LNCaP prostate tumor cells could be detected by PSA RT-PCR when added to female urine. CONCLUSION We have established a sensitive method of detecting prostatic cells in ejaculate and urethral washings and shown that PSA RT-PCR is a reliable indicator of prostate cells in these samples. However, RT-PCR for PSA, PSM and Apoliprotein D were not useful for discriminating malignant from non-malignant prostate cells.

The role of MscL amphipathic N terminus indicates a blueprint for bilayer-mediated gating of mechanosensitive channels

The bacterial mechanosensitive channel MscL gates in response to membrane tension as a result of mechanical force transmitted directly to the channel from the lipid bilayer. MscL represents an excellent model system to study the basic biophysical principles of mechanosensory transduction. However, understanding of the essential structural components that transduce bilayer tension into channel gating remains incomplete. Here using multiple experimental and computational approaches, we demonstrate that the amphipathic N-terminal helix of MscL acts as a crucial structural element during tension-induced gating, both stabilizing the closed state and coupling the channel to the membrane. We propose that this may also represent a common principle in the gating cycle of unrelated mechanosensitive ion channels, allowing the coupling of channel conformation to membrane dynamics.

Studying mechanosensitive ion channels using liposomes

Mechanosensitive (MS) ion channels are the primary molecular transducers of mechanical force into electrical and/or chemical intracellular signals in living cells. They have been implicated in innumerable mechanosensory physiological processes including touch and pain sensation, hearing, blood pressure control, micturition, cell volume regulation, tissue growth, or cellular turgor control. Much of what we know about the basic physical principles underlying the conversion of mechanical force acting upon membranes of living cells into conformational changes of MS channels comes from studies of MS channels reconstituted into artificial liposomes. Using bacterial MS channels as a model, we have shown by reconstituting these channels into liposomes that there is a close relationship between the physico-chemical properties of the lipid bilayer and structural dynamics bringing about the function of these channels.

Bacterial Mechanosensitive Channels: Models for Studying Mechanosensory Transduction

SIGNIFICANCE Sensations of touch and hearing are manifestations of mechanical contact and air pressure acting on touch receptors and hair cells of the inner ear, respectively. In bacteria, osmotic pressure exerts a significant mechanical force on their cellular membrane. Bacteria have evolved mechanosensitive (MS) channels to cope with excessive turgor pressure resulting from a hypo-osmotic shock. MS channel opening allows the expulsion of osmolytes and water, thereby restoring normal cellular turgor and preventing cell lysis. RECENT ADVANCES As biological force-sensing systems, MS channels have been identified as the best examples of membrane proteins coupling molecular dynamics to cellular mechanics. The bacterial MS channel of large conductance (MscL) and MS channel of small conductance (MscS) have been subjected to extensive biophysical, biochemical, genetic, and structural analyses. These studies have established MscL and MscS as model systems for mechanosensory transduction. CRITICAL ISSUES In recent years, MS ion channels in mammalian cells have moved into focus of mechanotransduction research, accompanied by an increased awareness of the role they may play in the pathophysiology of diseases, including cardiac hypertrophy, muscular dystrophy, or Xerocytosis. FUTURE DIRECTIONS A recent exciting development includes the molecular identification of Piezo proteins, which function as nonselective cation channels in mechanosensory transduction associated with senses of touch and pain. Since research on Piezo channels is very young, applying lessons learned from studies of bacterial MS channels to establishing the mechanism by which the Piezo channels are mechanically activated remains one of the future challenges toward a better understanding of the role that MS channels play in mechanobiology.

Patch-clamp characterization of the MscS-like mechanosensitive channel from Silicibacter pomeroyi.

Based on sequence similarity, the sp7 gene product, MscSP, of the sulfur-compound-decomposing Gram-negative marine bacterium Silicibacter pomeroyi belongs to the family of MscS-type mechanosensitive channels. To investigate MscSP channel properties, we measured its response to membrane tension using the patch-clamp technique on either a heterologous expression system using giant spheroplasts of MJF465 Escherichia coli strain (devoid of mechanosensitive channels MscL, MscS, and MscK), or on purified MscSP protein reconstituted in azolectin liposomes. These experiments showed typical pressure-dependent gating properties of a stretch-activated channel with a current/voltage plot indicating a rectifying behavior and weak preference for anions similar to the MscS channel of E. coli. However, the MscSP channel exhibited functional differences with respect to conductance and desensitization behavior, with the most striking difference between the two channels being the lack of inactivation in MscSP compared with MscS. This seems to result from the fact that although MscSP has a Gly in an equivalent position to MscS (G113), a position that is critical for inactivation, MscSP has a Glu residue instead of an Asn in a position that was recently shown to allosterically influence MscS inactivation, N117. To our knowledge, this study describes the first electrophysiological characterization of an MscS-like channel from a marine bacterium belonging to sulfur-degrading α-proteobacteria.

Activation of the mechanosensitive ion channel MscL by mechanical stimulation of supported Droplet-Hydrogel bilayers

The droplet on hydrogel bilayer (DHB) is a novel platform for investigating the function of ion channels. Advantages of this setup include tight control of all bilayer components, which is compelling for the investigation of mechanosensitive (MS) ion channels, since they are highly sensitive to their lipid environment. However, the activation of MS ion channels in planar supported lipid bilayers, such as the DHB, has not yet been established. Here we present the activation of the large conductance MS channel of E. coli, (MscL), in DHBs. By selectively stretching the droplet monolayer with nanolitre injections of buffer, we induced quantifiable DHB tension, which could be related to channel activity. The MscL activity response revealed that the droplet monolayer tension equilibrated over time, likely by insertion of lipid from solution. Our study thus establishes a method to controllably activate MS channels in DHBs and thereby advances studies of MS channels in this novel platform.

The protective effect of osmoprotectant TMAO on bacterial mechanosensitive channels of small conductance MscS/MscK under high hydrostatic pressure.

Activity of the bacterial mechanosensitive channels of small conductance MscS/MscK of E. coli was investigated under high hydrostatic pressure (HHP) using the “flying-patch” patch-clamp technique. The channels were gated by negative pipette voltage and their open probability was measured at HHP of 0.1 to 80 MPa. The channel open probability decreased with increasing HHP. When the osmolyte methylamine N-oxide (TMAO) was applied to the cytoplasmic side of the inside-out excised membrane patches of E. coli giant spheroplasts the inhibitory effect of HHP on the channel activity was suppressed at pressures of up to 40 MPa. At 40 MPa and above the channel open probability decreased in a similar fashion with or without TMAO. Our study suggests that TMAO helps to counteract the effect of HHP up to 40 MPa on the MscS/MscK open state by “shielding” the cytoplasmic domain of the channels.