Transmembrane Complex Research Articles

Assembly of a mechanosensitive complex is required to initiate sprouting angiogenesis

Endothelial cells (ECs) must respond rapidly to a variety of extracellular cues to initiate sprouting events. We undertook the current study to better understand how mechanical forces (shear stress) combine with biochemical cues to promote sprout outgrowth in a defined 3D model of EC invasion, because while the effects of shear stress have been widely studied in atherosclerosis, the molecular events directed by mechanical forces in angiogenic sprouting are incompletely understood. We initially focused on the known mechanosensitive protein Filamin A, which has been implicated in barrier regulation and vascular development. We found that Filamin A is required for EC sprouting in 3D and localizes to junctions with EC activation. Filamin A formed a complex with vascular endothelial (VE)‐cadherin and platelet endothelial cell adhesion molecule‐1 (PECAM‐1), two known members of mechanosensitive complex, following activation with shear stress and pro‐angiogenic factors. Silencing of Filamin A also disrupted the VE‐cadherin and PECAM‐1 complex, which correlates with decreased sprouting. Filamin A co‐localized with SHP2, a phosphatase previously implicated in barrier formation, following EC activation. SHP2 complexed with junctional molecules Filamin A, VE‐cadherin, and PECAM‐1, and complex formation between SHP2, VE‐cadherin, and PECAM‐1 was lost with Filamin A knockdown. In addition, in the absence of Filamin A expression, SHP2 failed to localize to endothelial junctions. Further, SHP2 silencing significantly reduced sprouting, and defects in sprouting were rescued by delivery of wild‐type SHP2. Altogether these data suggest that Filamin A recruits SHP2 to reinforce a transmembrane complex containing VE‐cadherin and PECAM‐1 that is needed to initiate EC sprouting. These data show for the first time that the formation of this mechanosensitive complex containing VE‐cadherin, PECAM‐1, Filamin A, and SHP2 is needed for successful sprout initiation.Support or Funding InformationWork supported by NIH RO1 HL095786, the Department of Molecular & Cellular Medicine, Texas A&M Health Science Center, and AHA Grant‐in‐Aid, Southwest Affiliate.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Conformational Dynamics of Sensory Rhodopsin II in Nanolipoprotein and Styrene-Maleic Acid Lipid Particles.

Styrene-maleic acid lipid particles (SMALPs) provide stable water-soluble nanocontainers for lipid-encased membrane proteins. Possible effects of the SMA-stabilized lipid environment on the interaction dynamics between functionally coupled membrane proteins remain to be elucidated. The photoreceptor sensory rhodopsin II, NpSRII and its cognate transducer, NpHtrII, of Natronomonas pharaonis form a transmembrane complex, NpSRII2 /NpHtrII2 that plays a key role in negative phototaxis and provides a unique model system to study the light-induced transfer of a conformational signal between two integral membrane proteins. Photon absorption induces transient structural changes in NpSRII comprising an outward movement of helix F that cause further conformational alterations in NpHtrII. We applied site-directed spin labeling and time-resolved optical and EPR spectroscopy to compare the conformational dynamics of NpSRII2 /NpHtrII2 reconstituted in SMALPs with that of nanolipoprotein particle and liposome preparations. NpSRII and NpSRII2 /NpHtrII2 show similar photocycles in liposomes and nanolipoprotein particles. An accelerated decay of the M photointermediate found for SMALPs can be explained by a high local proton concentration provided by the carboxylic groups of the SMA polymer. Light-induced large-scale conformational changes of NpSRII2 /NpHtrII2 observed in liposomes and nanolipoprotein particles are affected in SMALPs, indicating restrictions of the protein's conformational freedom.

Orientational Dynamics of Transition Dipoles and Exciton Relaxation in LH2 from Ultrafast Two-Dimensional Anisotropy.

Light-harvesting complexes in photosynthetic organisms display fast and efficient energy transfer dynamics, which depend critically on the electronic structure of the coupled chromophores within the complexes and their interactions with their environment. We present ultrafast anisotropy dynamics, resolved in both time and frequency, of the transmembrane light-harvesting complex LH2 from Rhodobacter sphaeroides in its native membrane environment using polarization-controlled two-dimensional electronic spectroscopy. Time-dependent anisotropy obtained from both experiment and modified Redfield simulation reveals an orientational preference for excited state absorption and an ultrafast equilibration within the B850 band in LH2. This ultrafast equilibration is favorable for subsequent energy transfer toward the reaction center. Our results also show a dynamic difference in excited state absorption anisotropy between the directly excited B850 population and the population that is initially excited at 800 nm, suggesting absorption from B850 states to higher-lying excited states following energy transfer from B850*. These results give insight into the ultrafast dynamics of bacterial light harvesting and the excited state energy landscape of LH2 in the native membrane environment.

Assembly and Post-assembly Turnover and Dynamics in the Type III Secretion System.

The type III secretion system (T3SS) is one of the largest transmembrane complexes in bacteria, comprising several intricately linked and embedded substructures. The assembly of this nanomachine is a hierarchical process which is regulated and controlled by internal and external cues at several critical points. Recently, it has become obvious that the assembly of the T3SS is not a unidirectional and deterministic process, but that parts of the T3SS constantly exchange or rearrange. This article aims to give an overview on the assembly and post-assembly dynamics of the T3SS, with a focus on emerging general concepts and adaptations of the general assembly pathway.

Structure of the type VI secretion system TssK\u2013TssF\u2013TssG baseplate subcomplex revealed by cryo-electron microscopy

Type VI secretion systems (T6SSs) translocate effectors into target cells and are made of a contractile sheath and a tube docked onto a multi-protein transmembrane complex via a baseplate. Although some information is available about the mechanisms of tail contraction leading to effector delivery, the detailed architecture and function of the baseplate remain unknown. Here, we report the 3.7 Å resolution cryo-electron microscopy reconstruction of an enteroaggregative Escherichia coli baseplate subcomplex assembled from TssK, TssF and TssG. The structure reveals two TssK trimers interact with a locally pseudo-3-fold symmetrical complex comprising two copies of TssF and one copy of TssG. TssF and TssG are structurally related to each other and to components of the phage T4 baseplate and of the type IV secretion system, strengthening the evolutionary relationships among these macromolecular machines. These results, together with bacterial two-hybrid assays, provide a structural framework to understand the T6SS baseplate architecture.

Genome-wide survey of efflux pump-coding genes associated with Cronobacter survival, osmotic adaptation, and persistence

Microorganisms express various transmembrane complexes collectively known as efflux pumps that influence their survival under stressful growth conditions. Although efflux pumps are important in resistance to antibiotics, disinfectants, and preservatives, little information about their presence and roles in Cronobacter spp. is available. To better understand the phylogeny, prevalence, and distribution of efflux pumps among strains of Cronobacter spp., whole genome sequencing (WGS) and pan-genomic DNA microarray (MA) analysis were carried out. Targeted MA analysis with 156 probe set alleles representing efflux pump genetic loci showed that all seven Cronobacter species possessed species-specific efflux pump orthologues. Distribution of the efflux pump genes and sequence variations were studied using WGS datasets. Phylogenetic and molecular clock analyses showed that the acquisition of these efflux pump genes possibly occurred at separate evolutionary events, which most likely correlated with the evolution of sequence type (ST) lineages. The acquisition of efflux pump genes either occurred as independent evolutionary events through horizontal gene transfer, or in certain situations, such as that for ST83 and ST4 strains, they could have been acquired through a robust microevolutionary selective process, some of which may have provided functional advantages. Efflux pump activity is very important in osmotic adaptation, survival, and persistence of Cronobacter species and are involved in several important physiological processes including protein, heavy metal, and sugar efflux. Understanding the mechanisms of Cronobacter adaptation in a wide range of environments, could eventually lead to development of methods to control the contamination of foods by these organisms.

Connecting the Dots between Mechanosensitive Channel Abundance, Osmotic Shock, and Survival at Single-Cell Resolution.

Rapid changes in extracellular osmolarity are one of many insults microbial cells face on a daily basis. To protect against such shocks, Escherichia coli and other microbes express several types of transmembrane channels that open and close in response to changes in membrane tension. In E. coli, one of the most abundant channels is the mechanosensitive channel of large conductance (MscL). While this channel has been heavily characterized through structural methods, electrophysiology, and theoretical modeling, our understanding of its physiological role in preventing cell death by alleviating high membrane tension remains tenuous. In this work, we examine the contribution of MscL alone to cell survival after osmotic shock at single-cell resolution using quantitative fluorescence microscopy. We conducted these experiments in an E. coli strain which is lacking all mechanosensitive channel genes save for MscL, whose expression was tuned across 3 orders of magnitude through modifications of the Shine-Dalgarno sequence. While theoretical models suggest that only a few MscL channels would be needed to alleviate even large changes in osmotic pressure, we find that between 500 and 700 channels per cell are needed to convey upwards of 80% survival. This number agrees with the average MscL copy number measured in wild-type E. coli cells through proteomic studies and quantitative Western blotting. Furthermore, we observed zero survival events in cells with fewer than ∼100 channels per cell. This work opens new questions concerning the contribution of other mechanosensitive channels to survival, as well as regulation of their activity.IMPORTANCE Mechanosensitive (MS) channels are transmembrane protein complexes which open and close in response to changes in membrane tension as a result of osmotic shock. Despite extensive biophysical characterization, the contribution of these channels to cell survival remains largely unknown. In this work, we used quantitative video microscopy to measure the abundance of a single species of MS channel in single cells, followed by their survival after a large osmotic shock. We observed total death of the population with fewer than ∼100 channels per cell and determined that approximately 500 to 700 channels were needed for 80% survival. The number of channels we found to confer nearly full survival is consistent with the counts of the numbers of channels in wild-type cells in several earlier studies. These results prompt further studies to dissect the contribution of other channel species to survival.

Mechanosensing at Cellular Interfaces.

At the plasma membrane interface, cells use various adhesions to sense their extracellular environment. These adhesions facilitate the transmission of mechanical signals that dictate cell behavior. This review discusses the mechanisms by which these mechanical signals are transduced through cell-matrix and cell-cell adhesions and how this mechanotransduction influences cell processes. Cell-matrix adhesions require the activation of and communication between various transmembrane protein complexes such as integrins. These links at the plasma membrane affect how a cell senses and responds to its matrix environment. Cells also communicate with each other through cell-cell adhesions, which further regulate cell behavior on a single- and multicellular scale. Coordination and competition between cell-cell and cell-matrix adhesions in multicellular aggregates can, to a significant extent, be modeled by differential adhesion analyses between the different interfaces even without knowing the details of cellular signaling. In addition, cell-matrix and cell-cell adhesions are connected by an intracellular cytoskeletal network that allows for direct communication between these distinct adhesions and activation of specific signaling pathways. Other membrane-embedded protein complexes, such as growth factor receptors and ion channels, play additional roles in mechanotransduction. Overall, these mechanoactive elements show the dynamic interplay between the cell, its matrix, and neighboring cells and how these relationships affect cellular function.

Autonomous control mechanism of stator assembly in the bacterial flagellar motor in response to changes in the environment.

The bacterial flagellar motor is composed of a rotor and a transmembrane ion channel complex that acts as a stator unit. The ion channel complex consists of at least three structural parts: a cytoplasmic domain responsible for the interaction with the rotor, a transmembrane ion channel that forms a pathway for the transit of ions across the cytoplasmic membrane, and a peptidoglycan-binding (PGB) domain that anchors the stator unit to the peptidoglycan (PG) layer. A flexible linker connecting the ion channel and the PGB domain not only coordinates stator assembly with its ion channel activity but also controls the assembly of stator units to the motor in response to changes in the environment. When the ion channel complex encounters the rotor, the N-terminal portion of the PGB domain adopts a partially stretched conformation, allowing the PGB domain to reach and bind to the PG layer. The binding affinity of the PGB domain for the PG layer is affected by the force applied to its anchoring point and to the type of ionic energy source. In this review article, we will present current understanding of autonomous control mechanism of stator assembly in the bacterial flagellar motor.

Abstract 1219: Association between MICA polymorphisms, s-MICA levels, and pancreatic cancer risk in a population-based case-control study

Abstract Objectives: The immune system has been shown to play an important role in pancreatic cancer. Abnormal cells express the transmembrane major histocompatibility complex class I chain-related gene A (MICA) protein, which is recognized by receptors present on NK and cytotoxic T cells. However, pancreatic tumor cells release MICA protein in soluble form (called s-MICA) from the tumor surface and thus avoid immune surveillance. In our previous study, we showed that a higher serum concentration of the s-MICA protein was associated with increased pancreatic cancer risk. In the current study, we hypothesized that functional variants in the MICA gene are associated with higher circulating s-MICA levels and increased pancreatic cancer risk. We focused on the A5.1 MICA allele, as this allele encodes a MICA protein that is shorter than its normal counterpart and is more easily cleaved from the cell surface. Methods: MICA alleles and s-MICA levels were measured in 116 cases and 492 controls (n=608) in a population-based case-control study in Minnesota. Allele assignments were based on the number of repeat units in the amplified regions of the MICA gene, and s-MICA levels were measured by enzyme-linked immunoabsorbent assay. To address the non-normal distribution of s-MICA levels, general linear regression with a log link was used to assess mean s-MICA levels across MICA alleles. Unconditional logistic regression was used to calculate the odds ratio (OR) and 95% confidence intervals (CI) for pancreatic cancer after adjustment for age, sex, education, smoking status, alcohol consumption, and diabetes status. The analysis was restricted to Caucasians, who represented 96% of all study participants. Results: 452 participants (74%) possessed at least one copy of the A5.1 allele. After adjustment for confounders, participants with at least one copy of the MICA A5.1 allele were at an increased risk of pancreatic cancer (multivariable OR=2.00, 95% CI: 1.06-3.79), compared to participants who did not possess any A5.1 allele. Participants who had at least one copy of the A5.1 allele had 2.46 (95%CI: 2.36-2.56) times greater mean s-MICA levels than those without the A5.1 allele. These associations were consistent among pancreatic cancer cases and controls. Conclusions: Our study supports the role of the MICA A5.1 allele in impaired immune response, which may increase the risk of pancreatic cancer. Citation Format: Guillaume C. Onyeaghala, John Lane, Nathan Pankratz, Heather H. Nelson, Bharat Thyagarajan, Kristin E. Anderson, Anna E. Prizment. Association between MICA polymorphisms, s-MICA levels, and pancreatic cancer risk in a population-based case-control study [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1219.

The ER membrane insertase Get1/2 is required for efficient mitophagy in yeast

Mitophagy is an evolutionarily conserved autophagy pathway that selectively eliminates mitochondria to control mitochondrial quality and quantity. Although mitophagy is thought to be crucial for cellular homeostasis, how this catabolic process is regulated remains largely unknown. Here we demonstrate that mitophagy during prolonged respiratory growth is strongly impaired in yeast cells lacking Get1/2, a transmembrane complex mediating insertion of tail-anchored (TA) proteins into the endoplasmic reticulum (ER) membrane. Under the same conditions, loss of Get1/2 caused only slight defects in other types of selective and bulk autophagy. In addition, mitophagy and other autophagy-related processes are mostly normal in cells lacking Get3, a cytosolic ATP-driven chaperone that promotes delivery of TA proteins to the Get1/2 complex. We also found that Get1/2-deficient cells exhibited wildtype-like induction and mitochondrial localization of Atg32, a protein essential for mitophagy. Notably, Get1/2 is important for Atg32-independent, ectopically promoted mitophagy. Together, we propose that Get1/2-dependent TA protein(s) and/or the Get1/2 complex itself may act specifically in mitophagy.

Distinct Electron Conductance Regimes in Bacterial Decaheme Cytochromes

AbstractShewanella oneidensis MR‐1 gains energy by extracellular electron transfer to solid surfaces. They employ c‐type cytochromes in two Mtr transmembrane complexes, forming a multiheme wire for electron transport across the cellular outer membrane. We investigated electron‐ and hole‐transfer mechanisms in the external terminal of the two complexes, MtrC and MtrF. Comparison of computed redox potentials with previous voltammetry experiments in distinct environments (isolated and electrode‐bound conditions of PFV or in vivo) suggests that these systems function in different regimes depending on the environment. Analysis of redox potential shifts in different regimes indicates strong coupling between the hemes via an interplay between direct Coulomb and indirect interactions through local structural reorganization. The latter results in the screening of Coulomb interactions and explains poor correlation of the strength of the heme‐to‐heme interactions with the distance between the hemes.

Distinct Electron Conductance Regimes in Bacterial Decaheme Cytochromes.

Shewanella oneidensis MR-1 gains energy by extracellular electron transfer to solid surfaces. They employ c-type cytochromes in two Mtr transmembrane complexes, forming a multiheme wire for electron transport across the cellular outer membrane. We investigated electron- and hole-transfer mechanisms in the external terminal of the two complexes, MtrC and MtrF. Comparison of computed redox potentials with previous voltammetry experiments in distinct environments (isolated and electrode-bound conditions of PFV or in vivo) suggests that these systems function in different regimes depending on the environment. Analysis of redox potential shifts in different regimes indicates strong coupling between the hemes via an interplay between direct Coulomb and indirect interactions through local structural reorganization. The latter results in the screening of Coulomb interactions and explains poor correlation of the strength of the heme-to-heme interactions with the distance between the hemes.

Structural modeling of an outer membrane electron conduit from a metal-reducing bacterium suggests electron transfer via periplasmic redox partners

Many subsurface microorganisms couple their metabolism to the reduction or oxidation of extracellular substrates. For example, anaerobic mineral-respiring bacteria can use external metal oxides as terminal electron acceptors during respiration. Porin–cytochrome complexes facilitate the movement of electrons generated through intracellular catabolic processes across the bacterial outer membrane to these terminal electron acceptors. In the mineral-reducing model bacterium Shewanella oneidensis MR-1, this complex is composed of two decaheme cytochromes (MtrA and MtrC) and an outer-membrane β-barrel (MtrB). However, the structures and mechanisms by which porin–cytochrome complexes transfer electrons are unknown. Here, we used small-angle neutron scattering (SANS) to study the molecular structure of the transmembrane complexes MtrAB and MtrCAB. Ab initio modeling of the scattering data yielded a molecular envelope with dimensions of ∼105 × 60 × 35 Å for MtrAB and ∼170 × 60 × 45 Å for MtrCAB. The shapes of these molecular envelopes suggested that MtrC interacts with the surface of MtrAB, extending ∼70 Å from the membrane surface and allowing the terminal hemes to interact with both MtrAB and an extracellular acceptor. The data also reveal that MtrA fully extends through the length of MtrB, with ∼30 Å being exposed into the periplasm. Proteoliposome models containing membrane-associated MtrCAB and internalized small tetraheme cytochrome (STC) indicate that MtrCAB could reduce Fe(III) citrate with STC as an electron donor, disclosing a direct interaction between MtrCAB and STC. Taken together, both structural and proteoliposome experiments support porin–cytochrome–mediated electron transfer via periplasmic cytochromes such as STC.

Assisted Insertion of Pseudomonas aeruginosa Type Three Secretion Translocator PopD in Membranes by Translocator PopB

Type III secretion (T3S) system is deployed by a wide range of pathogens to manipulate host cell response and establish infection. The T3S system is a syringe‐like apparatus that spans across the double membrane of bacteria, protruding 40nm–80nm into the extracellular space and connecting with target cell membrane. In Pseudomonas aeruginosa, the proteins PopB and PopD are secreted and found associated with the target eukaryotic cell membrane. These two proteins are believed to form a transmembrane complex or translocon to allow effector protein translocation. We have recently determined that PopD alone can spontaneously oligomerize and form mostly homo‐complexes of six subunits in the membrane. Interestingly, PopB and PopD together form membrane‐inserted hetero‐complexes that contain eight subunits of PopB and eight of PopD. However, the assembly mechanism to form these complexes remains unknown.Here, we investigated how PopD interacts with membranes when assembled into the PopB/D hetero‐complex using a combination of biochemical and biophysical techniques that report on the environment and location of individual amino acid. Our data suggested that the presence of PopB triggers conformational change in membrane associated PopD in lipid bilayers. Using a cell based phosphorylation assay, we also found a similar conformational change of PopD in infected HeLa cell membranes. Together, our results showed that PopB assists the membrane insertion of PopD.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Arabidopsis RETICULON-LIKE3 (RTNLB3) and RTNLB8 Participate in Agrobacterium-Mediated Plant Transformation.

Agrobacterium tumefaciens can genetically transform various eukaryotic cells because of the presence of a resident tumor-inducing (Ti) plasmid. During infection, a defined region of the Ti plasmid, transfer DNA (T-DNA), is transferred from bacteria into plant cells and causes plant cells to abnormally synthesize auxin and cytokinin, which results in crown gall disease. T-DNA and several virulence (Vir) proteins are secreted through a type IV secretion system (T4SS) composed of T-pilus and a transmembrane protein complex. Three members of Arabidopsis reticulon-like B (RTNLB) proteins, RTNLB1, 2, and 4, interact with VirB2, the major component of T-pilus. Here, we have identified that other RTNLB proteins, RTNLB3 and 8, interact with VirB2 in vitro. Root-based A. tumefaciens transformation assays with Arabidopsis rtnlb3, or rtnlb5-10 single mutants showed that the rtnlb8 mutant was resistant to A. tumefaciens infection. In addition, rtnlb3 and rtnlb8 mutants showed reduced transient transformation efficiency in seedlings. RTNLB3- or 8 overexpression transgenic plants showed increased susceptibility to A. tumefaciens and Pseudomonas syringae infection. RTNLB1-4 and 8 transcript levels differed in roots, rosette leaves, cauline leaves, inflorescence, flowers, and siliques of wild-type plants. Taken together, RTNLB3 and 8 may participate in A. tumefaciens infection but may have different roles in plants.

Members of Glycosyl-Hydrolase Family 17 of A. fumigatus Differentially Affect Morphogenesis.

Cell wall biosynthesis and remodeling are essential for fungal growth and development. In the fungal pathogen Aspergillus fumigatus, the β(1,3)glucan is the major cell wall polysaccharide. This polymer is synthesized at the plasma membrane by a transmembrane complex, then released into the parietal space to be remodeled by enzymes, and finally incorporated into the pre-existing cell wall. In the Glycosyl-Hydrolases family 17 (GH17) of A. fumigatus, two β(1,3)glucanosyltransferases, Bgt1p and Bgt2p, have been previously characterized. Disruption of BGT1 and BGT2 did not result in a phenotype, but sequence comparison and hydrophobic cluster analysis showed that three other genes in A. fumigatus belong to the GH17 family, SCW4, SCW11, and BGT3. In constrast to Δbgt1bgt2 mutants, single and multiple deletion of SCW4, SCW11, and BGT3 showed a decrease in conidiation associated with a higher conidial mortality and an abnormal conidial shape. Moreover, mycelium was also affected with a slower growth, stronger sensitivity to cell wall disturbing agents, and altered cell wall composition. Finally, the synthetic interactions between Bgt1p, Bgt2p, and the three other members, which support a functional cooperation in cell-wall assembly, were analyzed. Our data suggest that Scw4p, Scw11p, and Bgt3p are essential for cell wall integrity and might have antagonistic and distinct functions to Bgt1p and Bgt2p.

Membrane Anchoring of α-Helical Proteins: Role of Tryptophan.

The function of membrane proteins relies on a defined orientation of protein relative to lipid. In apparent correlation to protein anchoring, tryptophan residues are enriched in the lipid headgroup region. To characterize the thermodynamic and structural basis of this relationship in α-helical membrane proteins, we examined the role of three conserved tryptophans in the folding of the heterodimeric integrin αIIbβ3 transmembrane (TM) complex in phospholipid bicelles and mammalian membranes. In the homogenous lipid environment of bicelles, tryptophan was replaceable by residues of distinct polarities. The appropriate polarity was guided by the electrostatic potential of the tryptophan surrounding, suggesting that tryptophan can complement diverse environments by adjusting the orientation of its anisotropic side chain to achieve site-specific anchoring. As a sole membrane anchor, tryptophan made a contribution of 0.4 kcal/mol to TM complex stability in bicelles. In membranes, it proved more difficult to replace tryptophan even by tyrosine, indicating a superior capacity to interact with heterogeneous lipids of biological membranes. Interestingly, at intracellular TM helix ends, where integrin activation is initiated, sequence motifs that interact with lipids via opposing polarity patterns were found to restrict TM helix orientations beyond tryptophan anchoring. In contrast to bicelles, phenylalanine became the least accepted substitute in membranes, demonstrating an increased role of the hydrophobic effect. Altogether, our study implicates a wide amphiphilic range of tryptophan, membrane complexity, and the hydrophobic effect to be important factors in tryptophan membrane anchoring.

Morphological analysis of mitochondria for evaluating the toxicity of α-synuclein in transgenic mice and isolated preparations by atomic force microscopy

A key molecular event in the pathogenesis of Parkinson’s disease is mitochondrial damage caused by α-synuclein (α-syn). Mitochondria mediates both necrosis and apoptosis, which are associated with morphological changes. However, the mechanism by which α-syn alters mitochondrial morphology remains unclear. To address this issue, we investigated mitochondrial permeability transition pore (mPTP) opening and changes in cardiolipin (CL) levels in mitochondria isolated from the brain of Thy1α-syn mice. Cytoplasmic cytochrome C and cleaved caspase-3 protein levels were upregulated in the brain of transgenic mice. Morphological analysis by atomic force microscopy (AFM) suggested a correlation between mitochondrial morphology and function in these animals. Incubation of isolated mitochondria with recombinant human α-synuclein N terminus (α-syn/N) decreased mitochondrial CL content. An AFM analysis showed that α-syn/N induced mitochondrial swelling and the formation of pore-like structures, which was associated with decreased mitochondrial transmembrane potential and complex I activity. The observed mitochondrial dysfunction was abrogated by treatment with the mPTP inhibitor cyclosporin A, although there was no recovery of CL content. These results provide insight into the mechanism by which α-syn/N directly undermines mitochondrial structure and function via modulation of mPTP opening and CL levels, and suggests that morphological analysis of isolated mitochondria by AFM is a useful approach for evaluating mitochondrial injury.

Tail-Anchored Protein Insertion by a Single Get1/2 Heterodimer

The Get1/2 transmembrane complex drives the insertion of tail-anchored (TA) proteins from the cytosolic chaperone Get3 into the endoplasmic reticulum membrane. Mechanistic insight into how Get1/2 coordinates this process is confounded by a lack of understanding of the basic architecture of the complex. Here, we define the oligomeric state of full-length Get1/2 in reconstituted lipid bilayers by combining single-molecule and bulk fluorescence measurements with quantitative invitro insertion analysis. We show that a single Get1/2 heterodimer is sufficient for insertion and demonstrate that the conserved cytosolic regions of Get1 and Get2 bind asymmetrically to opposing subunits of the Get3 homodimer. Altogether, our results define a simplified model for how Get1/2 and Get3 coordinate TA protein insertion.