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The ability to sense and respond to osmotic fluctuations is critical for the maintenance of cellular integrity. We used gene co-essentiality analysis to identify an unappreciated relationship between TSC22D2, WNK1, and NRBP1 in regulating cell volume homeostasis. All of these genes have paralogs and are functionally buffered for osmo-sensing and cell volume control. Within seconds of hyperosmotic stress, TSC22D, WNK, and NRBP family members physically associate into biomolecular condensates, a process that is dependent on intrinsically disordered regions (IDRs). A close examination of these protein families across metazoans revealed that TSC22D genes evolved alongside a domain in NRBPs that specifically binds to TSC22D proteins, which we have termed NbrT (NRBP binding region with TSC22D), and this co-evolution is accompanied by rapid IDR length expansion in WNK-family kinases. Our study reveals that TSC22D, WNK, and NRBP genes evolved in metazoans to co-regulate rapid cell volume changes in response to osmolarity., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Mitochondrial morphology is an indicator of cellular health and function; however, its quantification and categorization into different subclasses is a complicated process. Here, we present a protocol for mitochondrial morphology quantification in the presence and absence of carbonyl cyanide m-chlorophenyl hydrazone stress. We describe steps for the preparation of cells for immunofluorescence microscopy, staining, and morphology quantification. The quantification protocol generates an aspect ratio that helps to categorize mitochondria into two clear subclasses. For complete details on the use and execution of this protocol, please refer to Nag et al. 1 ., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Mitochondrial morphology is regulated by the post-translational modifications of the dynamin family GTPase proteins including mitofusin 1 (MFN1), MFN2, and dynamin-related protein 1 (DRP1). Mitochondrial phosphatase phosphoglycerate mutase 5 (PGAM5) is emerging as a regulator of these post-translational modifications; however, its precise role in the regulation of mitochondrial morphology is unknown. We show that PGAM5 interacts with MFN2 and DRP1 in a stress-sensitive manner. PGAM5 regulates MFN2 phosphorylation and consequently protects it from ubiquitination and degradation. Further, phosphorylation and dephosphorylation modification of MFN2 regulates its fusion ability. Phosphorylation enhances fission and degradation, whereas dephosphorylation enhances fusion. PGAM5 dephosphorylates MFN2 to promote mitochondrial network formation. Further, using a Drosophila genetic model, we demonstrate that the MFN2 homolog Marf and dPGAM5 are in the same biological pathway. Our results identify MFN2 dephosphorylation as a regulator of mitochondrial fusion and PGAM5 as an MFN2 phosphatase., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Amyloids are fibrillar protein superstructures that are commonly associated with diseases in humans and with physiological functions in various organisms. The precise mechanisms of amyloid formation remain to be elucidated. Surprisingly, we discovered that a bacterial Escherichia coli chaperone-like ATPase, regulatory ATPase variant A (RavA), and specifically the LARA domain in RavA, forms amyloids under acidic conditions at elevated temperatures. RavA is involved in modulating the proper assembly of membrane respiratory complexes. LARA contains an N-terminal loop region followed by a β-sandwich-like folded core. Several approaches, including nuclear magnetic resonance spectroscopy and molecular dynamics simulations, were used to determine the mechanism by which LARA switches to an amyloid state. These studies revealed that the folded core of LARA is amyloidogenic and is protected by its N-terminal loop. At low pH and high temperatures, the interaction of the N-terminal loop with the folded core is disrupted, leading to amyloid formation., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

We use all-atom molecular dynamics simulations on a massive scale to compute the standard binding free energy of the 13-residue antimicrobial peptide indolicidin to a lipid bilayer. The analysis of statistical convergence reveals systematic sampling errors that correlate with reorganization of the bilayer on the microsecond timescale and persist throughout a total of 1.4 ms of sampling. Consistent with experimental observations, indolicidin induces membrane thinning, although the simulations significantly overestimate the lipophilicity of the peptide., (Copyright © 2014 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

Direct visualization of the mechanism(s) by which peptides induce localized changes to the structure of membranes has high potential for enabling understanding of the structure-function relationship in antimicrobial and cell-penetrating peptides. We have applied a combined imaging strategy to track the interaction of a model antimicrobial peptide, PFWRIRIRR-amide, with bacterial membrane-mimetic supported phospholipid bilayers comprised of POPE/TOCL. Our in situ studies revealed rapid reorganization of the POPE/TOCL membrane into localized TOCL-rich domains with a concomitant change in the organization of the membranes themselves, as reflected by changes in fluorescent-membrane-probe order parameter, upon introduction of the peptide., (2010 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

In situ atomic force microscopy (AFM) is an exceedingly powerful and useful technique for characterizing the structure and assembly of proteins in real-time, in situ, and especially at model membrane interfaces, such as supported planar lipid bilayers. There remains, however, a fundamental challenge with AFM-based imaging. Conclusions are inferred based on morphological or topographical features. It is conventionally very difficult to use AFM to confirm specific molecular conformation, especially in the case of protein-membrane interactions. In this case, a protein may undergo subtle conformational changes upon insertion in the membrane that may be critical to its function. AFM lacks the ability to directly measure such conformational changes and can, arguably, only resolve features that are topographically distinct. To address these issues, we have developed a platform that integrates in situ AFM with attenuated total reflection-Fourier transform infrared (ATR-FTIR) spectroscopy. This combination of tools provides a unique means of tracking, simultaneously, conformational changes, not resolvable by in situ AFM, with topographical details that are not readily identified by conventional spectroscopy. Preliminary studies of thermal transitions in supported lipid bilayers and direct evidence of lipid-induced conformational changes in adsorbed proteins illustrates the potential of this coupled in situ functional imaging strategy.

Determining the local structure, dynamics, and conformational requirements for protein-protein and protein-lipid interactions in membranes is critical to understanding biological processes ranging from signaling to the translocating and membranolytic action of antimicrobial peptides. We report here the application of a combined polarized total internal reflection fluorescence microscopy-in situ atomic force microscopy platform. This platform's ability to image membrane orientational order was demonstrated on DOPC/DSPC/cholesterol model membranes containing the fluorescent membrane probe, DiI-C(20) or BODIPY-PC. Spatially resolved order parameters and fluorophore tilt angles extracted from the polarized total internal reflection fluorescence microscopy images were in good agreement with the topographical details resolved by in situ atomic force microscopy, portending use of this technique for high-resolution characterization of membrane domain structures and peptide-membrane interactions.

Identifying the mechanisms responsible for the interaction of peptides with cell membranes is critical to the design of new antimicrobial peptides and membrane transporters. We report here the results of a computational simulation of the interaction of the 13-residue peptide indolicidin with single-phase lipid bilayers of dioleoylphosphatidylcholine, distearoylphosphatidylcholine, dioleoylphosphatidylglycerol, and distearoylphosphatidylglycerol. Ensemble analysis of the membrane-bound peptide revealed that, in contrast to the extended, linear backbone structure reported for indolicidin in sodium dodecyl sulphate detergent micelles, the peptide adopts a boat-shaped conformation in both phosphatidylglycerol and phosphatidylcholine lipid bilayers, similar to that reported for dodecylphosphocholine micelles. In agreement with fluorescence and NMR experiments, simulations confirmed that the peptide localizes in the membrane interface, with the distance between phosphate headgroups of each leaflet being reduced in the presence of indolicidin. These data, along with a concomitant decrease in lipid order parameters for the upper-tail region, suggest that indolicidin binding results in membrane thinning, consistent with recent in situ atomic force microscopy studies.

Despite the considerable information available with regards to the structure of the clostridial neurotoxins, and their inherent threat as biological warfare agents, the mechanisms underpinning their interactions with and translocation through the cell membrane remain poorly understood. We report herein the results of an in situ scanning probe microscopy study of the interaction of tetanus toxin C-fragment (Tet C) with supported planar lipid bilayers containing the ganglioside receptor G(T1b). Our results show that Tet C preferentially binds to the surface of fluid phase domains within biphasic membranes containing G(T1b) and that with an extended incubation period these interactions lead to dramatic changes in the morphology of the lipid bilayer, including the formation of 40-80 nm diameter circular cavities. Combined atomic force microscopy/total internal reflection fluorescence microscopy experiments confirmed the presence of Tet C in the membrane after extended incubation. These morphological changes were found to be dependent upon the presence of G(T1b) and the solution pH.

Glucagon solutions at pH 2.0 were subjected to mechanical agitation at 37 degrees C in the presence of a hydrophobic surface to explore the details of aggregation and fiber formation. High-resolution intermittent-contact atomic force microscopy performed in solution revealed the presence of aggregates after 0.5 h; however, longer agitation times resulted in the formation of fibrillated structures with varying levels of higher-order assembly. Height, periodicity, and amplitude measurements of these structures allowed the identification of four distinct fiber types. The most elementary fiber form, designated a filament, self-associates in a specific wound fashion to produce protofibrils composed of two filaments. Subsequent self-assembly of these filaments and protofibrils leads to two well-defined fibrillar motifs, termed Type I and Type II. Atomic force microscopy imaging of pH 2.8 glucagon solutions not agitated or exposed to elevated temperature revealed the presence of amorphous aggregates before the formation of fibrillar structures similar to those seen at pH 2.0. Time-course solution Fourier transform infrared spectroscopy and thioflavin T binding studies suggested that glucagon aggregation and fibril formation were associated with the development of beta-sheet structure. The results of these studies are used to describe a possible mechanism for glucagon aggregation and fibrillation that is consistent with a hierarchical assembly model proposed for amyloid fibril formation.

Coupling atomic force microscopy (AFM) with high-resolution fluorescence microscopy is an attractive means of identifying membrane domains by both physical topography and fluorescence. We have used this approach to study the ability of a suite of fluorescent molecules to probe domain structures in supported planar bilayers. These included BODIPY-labeled ganglioside, sphingomyelin, and three new cholesterol derivatives, as well as NBD-labeled phosphatidylcholine, sphingomyelin, and cholesterol. Interestingly, many fluorescent lipid probes, including derivatives of known raft-associated lipids, preferentially partitioned into topographical features consistent with nonraft domains. This suggests that the covalent attachment of a small fluorophore to a lipid molecule can abolish its ability to associate with rafts. In addition, the localization of one of the BODIPY-cholesterol derivatives was dependent on the lipid composition of the bilayer. These data suggest that conclusions about the identification of membrane domains in supported planar bilayers on the basis of fluorescent lipid probes alone must be interpreted with caution. The combination of AFM with fluorescence microscopy represents a more rigorous means of identifying lipid domains in supported bilayers.

Identifying the mechanisms responsible for the assembly of proteins into higher-order structures is fundamental to structural biology and understanding specific disease pathways. The amyloid-beta (Abeta) peptide is illustrative in this regard as fibrillar deposits of Abeta are characteristic of Alzheimer's disease. Because Abeta includes portions of the extracellular and transmembrane domains of the amyloid precursor protein, it is crucial to understand how this peptide interacts with cell membranes and specifically the role of membrane structure and composition on Abeta assembly and cytotoxicity. We describe the results of a combined circular dichroism spectroscopy, electron microscopy, and in situ tapping mode atomic force microscopy (TMAFM) study of the interaction of soluble monomeric Abeta with planar bilayers of total brain lipid extract. In situ extended-duration TMAFM provided evidence of membrane disruption via fibril growth of initially monomeric Abeta1-40 peptide within the total brain lipid bilayers. In contrast, the truncated Abeta1-28 peptide, which lacks the anchoring transmembrane domain found in Abeta1-40, self-associates within the lipid headgroups but does not undergo fibrillogenesis. These observations suggest that the fibrillogenic properties of Abeta peptide are in part a consequence of membrane composition, peptide sequence, and mode of assembly within the membrane.

Crystallographic studies of insulin-protamine complexes, such as neutral protamine Hagedorn (NPH) insulin, have been hampered by high crystal solvent content, small crystal dimensions, and extensive disorder in the protamine molecules. We report herein in situ tapping mode atomic force microscopy (TMAFM) studies of crystalline neutral protamine Lys(B28)Pro(B29) (NPL), a complex of Lys(B28)Pro(B29) insulin, in which the C-terminal prolyl and lysyl residues of human insulin are inverted, and protamine that is used as an intermediate time-action therapy for treating insulin-dependent diabetes. Tapping mode AFM performed at 6 degrees C on bipyramidally tipped tetragonal rod-shaped NPL crystals revealed large micron-sized islands separated by 44-A tall steps. Lattice images obtained by in situ TMAFM phase and height imaging on these islands were consistent with the arrangement of individual insulin-protamine complexes on the P4(1)2(1)2 (110) crystal plane of NPH, based on a low-resolution x-ray diffraction structure of NPH, arguing that the NPH and NPL insulins are isostructural. Superposition of the height and phase images indicated that tip-sample adhesion was larger in the interstices between NPL complexes in the (110) crystal plane than over the individual complexes. These results demonstrate the utility of low-temperature TMAFM height and phase imaging for the structural characterization of biomolecular complexes.

Although x-ray crystal structures exist for many forms of insulin, the hormone involved in glucose metabolism and used in the treatment of diabetes, x-ray structural characterization of therapeutically important long-acting crystalline ultralente insulin forms has been elusive because of small crystal size and poor diffraction characteristics. We describe tapping-mode atomic force microscopy (TMAFM) studies, performed directly in crystallization liquor, of ultralente crystals prepared from bovine, human, and porcine insulins. Lattice images obtained from direct imaging of crystal planes are consistent with R3 space group symmetry for each insulin type, but the morphology of the human and porcine crystals observed by AFM differs substantially from that of the bovine insulin crystals. Human and porcine ultralente crystals exhibited large, molecularly flat (001) faces consisting of hexagonal arrays of close packed hexamers. In contrast, bovine ultralente crystals predominantly exhibited faces with cylindrical features assignable to close-packed stacks of insulin hexamers laying in-plane, consistent with the packing motif of the (010) and (011) planes. This behavior is attributed to a twofold increase in the hydrophobic character of the upper and lower surfaces of the donut-shaped insulin hexamer in bovine insulin compared to its human and porcine counterparts that results from minor sequence differences between these insulins. The increased hydrophobicity of these surfaces can promote hexamer-hexamer stacking in precrystalline aggregates or enhance attachment of single hexamers along the c axis at the crystal surface during crystal growth. Both events lead to enhanced growth of ¿hk0¿ planes instead of (001). The insulin hexamers on the (010) and (110) faces are exposed "edge-on" to the aqueous medium, such that solvent access to the center of the hexamer and to solvent channels is reduced compared to the (001) surface, consistent with the slower dissolution and reputed unique basal activity of bovine ultralente insulin. These observations demonstrate that subtle variations in amino acid sequence can dramatically affect the interfacial structure of crystalline proteins.

The self-association of proteins is influenced by amino acid sequence, molecular conformation, and the presence of molecular additives. In the presence of phenolic additives, LysB28ProB29 insulin, in which the C-terminal prolyl and lysyl residues of wild-type human insulin have been inverted, can be crystallized into forms resembling those of wild-type insulins in which the protein exists as zinc-complexed hexamers organized into well-defined layers. We describe herein tapping-mode atomic force microscopy (TMAFM) studies of single crystals of rhombohedral (R3) LysB28ProB29 that reveal the influence of sequence variation on hexamer-hexamer association at the surface of actively growing crystals. Molecular scale lattice images of these crystals were acquired in situ under growth conditions, enabling simultaneous identification of the rhombohedral LysB28ProB29 crystal form, its orientation, and its dynamic growth characteristics. The ability to obtain crystallographic parameters on multiple crystal faces with TMAFM confirmed that bovine and porcine insulins grown under these conditions crystallized into the same space group as LysB28ProB29 (R3), enabling direct comparison of crystal growth behavior and the influence of sequence variation. Real-time TMAFM revealed hexamer vacancies on the (001) terraces of LysB28ProB29, and more rounded dislocation noses and larger terrace widths for actively growing screw dislocations compared to wild-type bovine and porcine insulin crystals under identical conditions. This behavior is consistent with weaker interhexamer attachment energies for LysB28ProB29 at active growth sites. Comparison of the single crystal x-ray structures of wild-type insulins and LysB28ProB29 suggests that differences in protein conformation at the hexamer-hexamer interface and accompanying changes in interhexamer bonding are responsible for this behavior. These studies demonstrate that subtle changes in molecular conformation due to a single sequence inversion in a region critical for insulin self-association can have a significant effect on the crystallization of proteins.

Atomic force microscopy performed on single crystals of three different polymorphs of bovine insulin revealed molecularly smooth (001) layers separated by steps whose heights reflect the dimensions of a single insulin hexamer. Whereas contact mode imaging caused etching that prevented molecular-scale resolution, tapping mode imaging in solution provided molecular-scale contrast that enabled determination of lattice parameters and polymorph identification while simultaneously enabling real-time examination of growth modes and assessment of crystal quality. Crystallization proceeds layer by layer, a process in which the protein molecules assemble homoepitaxially with nearly perfect orientational and translational commensurism. Tapping mode imaging also revealed insulin aggregates attached to the (001) faces, their incorporation into growing terraces, and their role in defect formation. These observations demonstrate that tapping mode imaging is ideal for real-time in situ investigation of the crystallization of soft protein crystals of relatively small proteins such as insulin, which cannot withstand the lateral shear forces exerted by the scanning probe in conventional imaging modes.