Cell-cycle-dependent structural transitions in the human CENP-A nucleosome in vivo.

In eukaryotes, DNA is packaged into chromatin by canonical histone proteins. The specialized histone H3 variant CENP-A provides an epigenetic and structural basis for chromosome segregation by replacing H3 at centromeres. Unlike exclusively octameric canonical H3 nucleosomes, CENP-A nucleosomes have been shown to exist as octamers, hexamers, and tetramers. An intriguing possibility reconciling these observations is that CENP-A nucleosomes cycle between octamers and tetramers in vivo. We tested this hypothesis by tracking CENP-A nucleosomal components, structure, chromatin folding, and covalent modifications across the human cell cycle. We report that CENP-A nucleosomes alter from tetramers to octamers before replication and revert to tetramers after replication. These structural transitions are accompanied by reversible chaperone binding, chromatin fiber folding changes, and previously undescribed modifications within the histone fold domains of CENP-A and H4. Our results reveal a cyclical nature to CENP-A nucleosome structure and have implications for the maintenance of epigenetic memory after centromere replication.

A novel cysteine-rich adaptor protein is required for mucin packaging and secretory granule stability in vivo.

Mucins are large, highly glycosylated extracellular matrix proteins that line and protect epithelia of the respiratory, digestive, and urogenital tracts. Previous work has shown that mucins form large, interconnected polymeric networks that mediate their biological functions once secreted. However, how these large matrix molecules are compacted and packaged into much smaller secretory granules within cells prior to secretion is largely unknown. Here, we demonstrate that a small cysteine-rich adaptor protein is essential for proper packaging of a secretory mucin in vivo. This adaptor acts via cysteine bonding between itself and the cysteine-rich domain of the mucin. Loss of this adaptor protein disrupts mucin packaging in secretory granules, alters the mobile fraction within granules, and results in granules that are larger, more circular, and more fragile. Understanding the factors and mechanisms by which mucins and other highly glycosylated matrix proteins are properly packaged and secreted may provide insight into diseases characterized by aberrant mucin secretion.

A phage-encoded nucleoid associated protein compacts both host and phage DNA and derepresses H-NS silencing.

Nucleoid Associated Proteins (NAPs) organize the bacterial chromosome within the nucleoid. The interaction of the NAP H-NS with DNA also represses specific host and xenogeneic genes. Previously, we showed that the bacteriophage T4 early protein MotB binds to DNA, co-purifies with H-NS/DNA, and improves phage fitness. Here we demonstrate using atomic force microscopy that MotB compacts the DNA with multiple MotB proteins at the center of the complex. These complexes differ from those observed with H-NS and other NAPs, but resemble those formed by the NAP-like proteins CbpA/Dps and yeast condensin. Fluorescent microscopy indicates that expression of motB in vivo, at levels like that during T4 infection, yields a significantly compacted nucleoid containing MotB and H-NS. motB overexpression dysregulates hundreds of host genes; ∼70% are within the hns regulon. In infected cells overexpressing motB, 33 T4 late genes are expressed early, and the T4 early gene repEB, involved in replication initiation, is up ∼5-fold. We postulate that MotB represents a phage-encoded NAP that aids infection in a previously unrecognized way. We speculate that MotB-induced compaction may generate more room for T4 replication/assembly and/or leads to beneficial global changes in host gene expression, including derepression of much of the hns regulon.

Intrinsic elasticity of nucleosomes is encoded by histone variants and calibrated by their binding partners.

Histone variants fine-tune transcription, replication, DNA damage repair, and faithful chromosome segregation. Whether and how nucleosome variants encode unique mechanical properties to their cognate chromatin structures remains elusive. Here, using in silico and in vitro nanoindentation methods, extending to in vivo dissections, we report that histone variant nucleosomes are intrinsically more elastic than their canonical counterparts. Furthermore, binding proteins, which discriminate between histone variant nucleosomes, suppress this innate elasticity and also compact chromatin. Interestingly, when we overexpress the binding proteins in vivo, we also observe increased compaction of chromatin enriched for histone variant nucleosomes, correlating with diminished access. Taken together, these data suggest a plausible link between innate mechanical properties possessed by histone variant nucleosomes, the adaptability of chromatin states in vivo, and the epigenetic plasticity of the underlying locus.

Tyrosinase Nanoparticles: Understanding the Melanogenesis Pathway by Isolating the Products of Tyrosinase Enzymatic Reaction.

Human Tyrosinase (Tyr) is the rate-limiting enzyme of the melanogenesis pathway. Tyr catalyzes the oxidation of the substrate L-DOPA into dopachrome and melanin. Currently, the characterization of dopachrome-related products is difficult due to the absence of a simple way to partition dopachrome from protein fraction. Here, we immobilize catalytically pure recombinant human Tyr domain (residues 19-469) containing 6xHis tag to Ni-loaded magnetic beads (MB). Transmission electron microscopy revealed Tyr-MB were within limits of 168.2 ± 24.4 nm while the dark-brown melanin images showed single and polymerized melanin with a diameter of 121.4 ± 18.1 nm. Using Hill kinetics, we show that Tyr-MB has a catalytic activity similar to that of intact Tyr. The diphenol oxidase reactions of L-DOPA show an increase of dopachrome formation with the number of MB and with temperature. At 50 °C, Tyr-MB shows some residual catalytic activity suggesting that the immobilized Tyr has increased protein stability. In contrast, under 37 °C, the dopachrome product, which is isolated from Tyr-MB particles, shows that dopachrome has an orange-brown color that is different from the color of the mixture of L-DOPA, Tyr, and dopachrome. In the future, Tyr-MB could be used for large-scale productions of dopachrome and melanin-related products and finding a treatment for oculocutaneous albinism-inherited diseases.

Protein Stability and Functional Characterization of Intra-Melanosomal Domain of Human Recombinant Tyrosinase-Related Protein 1.

Pigmentation is the result of a complex process by which the biopolymer melanin is synthesized and packed into melanosomes of melanocytes. Various types of oculocutaneous albinism (OCA), a series of autosomal recessive disorders, are associated with reduced pigmentation in the skin, eyes, and hair due to genetic mutations of proteins involved in melanogenesis. Human tyrosinase (Tyr) and tyrosinase-related protein 1 (Tyrp1) drives the enzymatic process of pigment bio-polymerization. However, within the melanogenic pathway, Tyrp1 has catalytic functions not clearly defined and distinct from Tyr. Here, we characterize the biochemical and biophysical properties of recombinant human Tyrp1. For this purpose, we purified and analyzed the intra-melanosomal domain (Tyrp1tr) for protein stability and enzymatic function in conditions mimicking the environment within melanosomes and the endoplasmic reticulum. The study suggests that Tyrp1tr is a monomeric molecule at ambient temperatures and below (<25 °C). At higher temperatures, >31 °C, higher protein aggregates form with a concurrent decrease of monomers in solution. Also, Tyrp1tr diphenol oxidase activity at pH 5.5 rises as both the pre-incubation temperature and the higher molecular weight protein aggregates formation increases. The enhanced protein activity is consistent with the volume exclusion change caused by protein aggregates.

Durotaxis by Human Cancer Cells.

Durotaxis is a type of directed cell migration in which cells respond to a gradient of extracellular stiffness. Using automated tracking of positional data for large sample sizes of single migrating cells, we investigated 1) whether cancer cells can undergo durotaxis; 2) whether cell durotactic efficiency varies depending on the regional compliance of stiffness gradients; 3) whether a specific cell migration parameter such as speed or time of migration correlates with durotaxis; and 4) whether Arp2/3, previously implicated in leading edge dynamics and migration, contributes to cancer cell durotaxis. Although durotaxis has been characterized primarily in nonmalignant mesenchymal cells, little is known about its role in cancer cell migration. Diffusible factors are known to affect cancer cell migration and metastasis. However, because many tumor microenvironments gradually stiffen, we hypothesized that durotaxis might also govern migration of cancer cells. We evaluated the durotactic potential of multiple cancer cell lines by employing substrate stiffness gradients mirroring the physiological stiffness encountered by cells in a variety of tissues. Automated cell tracking permitted rapid acquisition of positional data and robust statistical analyses for migrating cells. These durotaxis assays demonstrated that all cancer cell lines tested (two glioblastoma, metastatic breast cancer, and fibrosarcoma) migrated directionally in response to changes in extracellular stiffness. Unexpectedly, all cancer cell lines tested, as well as noninvasive human fibroblasts, displayed the strongest durotactic migratory response when migrating on the softest regions of stiffness gradients (2-7 kPa), with decreased responsiveness on stiff regions of gradients. Focusing on glioblastoma cells, durotactic forward migration index and displacement rates were relatively stable over time. Correlation analyses showed the expected correlation with displacement along the gradient but much less with persistence and none with cell speed. Finally, we found that inhibition of Arp2/3, an actin-nucleating protein necessary for lamellipodial protrusion, impaired durotactic migration.

Macrophages Shed Excess Cholesterol in Unique Extracellular Structures Containing Cholesterol Microdomains.

OBJECTIVE: Cells use various mechanisms to maintain cellular cholesterol homeostasis including efflux of cholesterol from the cellular plasma membrane to cholesterol acceptors such as HDLs (high-density lipoproteins). Little is known about the transfer of cholesterol from cells into the extracellular matrix. Using a unique monoclonal antibody that detects ordered cholesterol arrays (ie, cholesterol micro[or nano]-domains), we previously identified that particles containing these cholesterol domains accumulate in the extracellular matrix during cholesterol enrichment of human monocyte-derived macrophages and are found in atherosclerotic lesions. In this study, we further investigate these deposited particles containing cholesterol microdomains and discover their unexpected morphology. APPROACH AND RESULTS: Although appearing spherical at the resolution of the conventional fluorescence microscope, super-resolution immunofluorescence and atomic force microscopy of in situ cholesterol microdomains, and immunoelectron microscopy of isolated cholesterol microdomains revealed that the microdomains are not vesicles or 3-dimensional crystals but rather appear as branching irregularly shaped deposits of varying size. These cholesterol microdomain-containing deposits are shed from the plasma membrane into the extracellular matrix. CONCLUSIONS: To date, research on cellular excretion of excess cholesterol has demonstrated cellular cholesterol efflux in the form of membranous vesicles and discoidal HDL particles released into the fluid-phase medium. Shedding of plasma membrane cholesterol microdomains provides an additional mechanism for cells such as macrophages to maintain plasma membrane cholesterol homeostasis. Furthermore, recognition that macrophages shed cholesterol microdomains into the extracellular matrix is important to our understanding of extracellular buildup of cholesterol in atherosclerosis.

Microscale mapping of extracellular matrix elasticity of mouse joint cartilage: an approach to extracting bulk elasticity of soft matter with surface roughness.

Cartilage is composed of cells and an extracellular matrix, the latter being a composite of a collagen mesh interpenetrated by proteoglycans responsible for tissue osmotic swelling. The matrix composition and structure vary through the tissue depth. Mapping such variability requires tissue sectioning to gain access. The resulting surface roughness, and concomitant proteoglycan loss contribute to large uncertainties in elastic modulus estimates. To extract elasticity values for the bulk matrix which are not obfuscated by the indeterminate surface layer, we developed a novel experimental and data analysis methodology. We analyzed the surface roughness to optimize the probe size, and performed high-resolution (1 µm) elasticity mapping on thin (∼12 µm), epiphyseal newborn mouse cartilage sections cut parallel to the bone longitudinal axis or normal to the articular surface. Mild fixation prevented the major proteoglycan loss observed in unfixed specimens but not the stress release that resulted in thickness changes in the sectioned matrix. Our novel data analysis method introduces a virtual contact point as a fitting parameter for the Hertz model, to minimize the effects of surface roughness and corrects for the finite section thickness. Our estimates of cartilage elasticity converge with increasing indentation depth and, unlike previous data interpretations, are consistent with linearly elastic material. A high cell density that leaves narrow matrix septa between cells may cause the underestimation of elastic moduli, whereas fixation probably causes an overestimation. The proposed methodology has broader relevance to nano- and micro-indentation of soft materials with multiple length scales of organization and whenever surface effects (including roughness, electrostatics, van der Waals forces, etc.) become significant.

Initial stages of V(D)J recombination: the organization of RAG1/2 and RSS DNA in the postcleavage complex.

To obtain structural information on the early stages of V(D)J recombination, we isolated a complex of the core RAG1 and RAG2 proteins with DNA containing a pair of cleaved recombination signal sequences (RSS). Stoichiometric and molecular mass analysis established that this signal-end complex (SEC) contains two protomers each of RAG1 and RAG2. Visualization of the SEC by negative-staining electron microscopy revealed an anchor-shaped particle with approximate two-fold symmetry. Consistent with a parallel arrangement of DNA and protein subunits, the N termini of RAG1 and RAG2 are positioned at opposing ends of the complex, and the DNA chains beyond the RSS nonamer emerge from the same face of the complex, near the RAG1 N termini. These first images of the V(D)J recombinase in its postcleavage state provide a framework for modeling RAG domains and their interactions with DNA.

Galactose repressor mediated intersegmental chromosomal connections in Escherichia coli.

By microscopic analysis of fluorescent-labeled GalR, a regulon-specific transcription factor in Escherichia coli, we observed that GalR is present in the cell as aggregates (one to three fluorescent foci per cell) in nongrowing cells. To investigate whether these foci represent GalR-mediated association of some of the GalR specific DNA binding sites (gal operators), we used the chromosome conformation capture (3C) method in vivo. Our 3C data demonstrate that, in stationary phase cells, many of the operators distributed around the chromosome are interacted. By the use of atomic force microscopy, we showed that the observed remote chromosomal interconnections occur by direct interactions between DNA-bound GalR not involving any other factors. Mini plasmid DNA circles with three or five operators positioned at defined loci showed GalR-dependent loops of expected sizes of the intervening DNA segments. Our findings provide unique evidence that a transcription factor participates in organizing the chromosome in a three-dimensional structure. We believe that these chromosomal connections increase local concentration of GalR for coordinating the regulation of widely separated target genes, and organize the chromosome structure in space, thereby likely contributing to chromosome compaction.

DNA nanoparticles with core-shell morphology.

Mannobiose-modified polyethylenimines (PEI) are used in gene therapy to generate nanoparticles of DNA that can be targeted to the antigen-presenting cells of the immune system. We report that the sugar modification alters the DNA organization within the nanoparticles from homogenous to shell-like packing. The depth-dependent packing of DNA within the nanoparticles was probed using AFM nano-indentation. Unmodified PEI-DNA nanoparticles display linear elastic properties and depth-independent mechanics, characteristic of homogenous materials. Mannobiose-modified nanoparticles, however, showed distinct force regimes that were dependent on indentation depth, with 'buckling'-like response that is reproducible and not due to particle failure. By comparison with theoretical studies of spherical shell mechanics, the structure of mannobiosylated particles was deduced to be a thin shell with wall thickness in the order of few nanometers, and a fluid-filled core. The shell-core structure is also consistent with observations of nanoparticle denting in altered solution conditions, with measurements of nanoparticle water content from AFM images, and with images of DNA distribution in Transmission Electron Microscopy.

HIV-1 Integrase Assembles Multiple Species of Stable Synaptic Complex Intasomes That Are Active for Concerted DNA Integration In vitro.

Retroviral DNA integration is mediated by nucleoprotein complexes (intasomes) in which a pair of viral DNA ends are bridged by a multimer of integrase (IN). Most of the high-resolution structures of HIV-1 intasomes are based on an HIV-1 IN with an Sso7d protein domain fused to the N-terminus. Sso7d-IN aggregates much less than wild-type IN and has been critical for structural studies of HIV-1 intasomes. Unexpectedly, these structures revealed that the common core architecture that mediates catalysis could be assembled in various ways, giving rise to both tetrameric and dodecameric intasomes, together with other less well-characterized species. This differs from related retroviruses that assemble unique multimeric intasomes, although the number of protomers in the intasome varies between viruses. The question of whether the additional Sso7d domain contributes to the heterogeneity of HIV-1 intasomes is therefore raised. We have addressed this by biochemical and structural studies of intasomes assembled with wild-type HIV-1 IN. Negative stain and cryo-EM reveal a similar range of multimeric intasome species as with Sso7d-IN with the same common core architecture. Stacks of intasomes resulting from domain swapping are also seen with both wild-type and Sso7d-IN intasomes. The propensity to assemble multimeric intasome species is, therefore, an intrinsic property of HIV-1 IN and is not conferred by the presence of the Sso7d domain. The recently solved intasome structures of different retroviral species, which have been reported to be tetrameric, octameric, dodecameric, and hexadecameric, highlight how a common intasome core architecture can be assembled in different ways for catalysis.

Tetrameric organization of vertebrate centromeric nucleosomes.

Mitosis ensures equal genome segregation in the eukaryotic lineage. This process is facilitated by microtubule attachment to each chromosome via its centromere. In centromeres, canonical histone H3 is replaced in nucleosomes by a centromere-specific histone H3 variant (CENH3), providing the unique epigenetic signature required for microtubule binding. Due to recent findings of alternative CENH3 nucleosomal forms in invertebrate centromeres, it has been debated whether the classical octameric nucleosomal arrangement of two copies of CENH3, H4, H2A, and H2B forms the basis of the vertebrate centromere. To address this question directly, we examined CENH3 [centromere protein A (CENP-A)] nucleosomal organization in human cells, using a combination of nucleosome component analysis, atomic force microscopy (AFM), and immunoelectron microscopy (immuno-EM). We report that native CENP-A nucleosomes contain centromeric alpha satellite DNA, have equimolar amounts of H2A, H2B, CENP-A, and H4, and bind kinetochore proteins. These nucleosomes, when measured by AFM, yield one-half the dimensions of canonical octameric nucleosomes. Using immuno-EM, we find that one copy of CENP-A, H2A, H2B, and H4 coexist in CENP-A nucleosomes, in which internal C-terminal domains are accessible. Our observations indicate that CENP-A nucleosomes are organized as asymmetric heterotypic tetramers, rather than canonical octamers. Such altered nucleosomes form a chromatin fiber with distinct folding characteristics, which we utilize to discriminate tetramers directly within bulk chromatin. We discuss implications of our observations in the context of universal epigenetic and mechanical requirements for functional centromeres.

Sulfated endospermic nanocellulose crystals prevent the transmission of SARS-CoV-2 and HIV-1.

Biomaterials with antimicrobial activity are gaining attention due to their biodegradability and efficacy in interacting with a wide range of microorganisms. A new cellulose nano-biomaterial, endospermic nanocellulose crystals (ENC) obtained from parenchymal tissue of ivory nut endosperm, has a natural capacity as a universal binder. This feature is enhanced when it is chemically functionalized, and can be exploited in the fight against microbes. We tested the ability of sulfated ENC in aqueous suspension to encapsulate viruses through a crosslinking reaction mediated by cations. 0.25% w/v ENC suspensions efficiently encapsulated spike (S) protein, preventing its interaction with ACE2 receptor. ENC was further able to encapsulate SARS-CoV-2 pseudoviruses and prevent infection of 293T-hsACE2 cells. ENC also suppressed infection of MT-4 cells with HIV-1LAI.04. This antiviral activity of sulfated ENC is due to the irreversible interaction of ENC with viral particles mediated by crosslinking, as antiviral activity was less effective in the absence of cations. Additionally, ENC was used as a matrix to immobilize recombinant ACE2 receptors and anti-S IgG, creating molecular lures that efficiently inhibited SARS-CoV-2 infections in vitro. These results show that sulfated ENC from ivory nuts can be used as an efficient antiviral material.

The linker of the interferon response factor 3 transcription factor is not unfolded.

Interferon response factor 3 (IRF-3) is a transcription factor that plays an essential role in controlling the synthesis of interferon-ß (IFN-ß) and is a protein consisting of two well-defined domains, the N-terminal DNA-binding and the C-terminal dimerization domains, connected by a 75-residue linker, supposedly unfolded. However, it was not clear whether in intact IRF-3 this linker segment of the chain, which carries the nuclear export signal and includes a region of high helical propensity, remains unfolded. This has been investigated using nuclear magnetic resonance by ligating the (15)N-labeled linker to the unlabeled N-terminal and C-terminal domains. It was found that, while the linker alone is indeed in a completely unfolded state, when ligated to the C-terminal domain it shows some ordering, and this ordering becomes much more pronounced when the linker is also ligated to the N-terminal domain. Thus, in intact IRF-3, the linker represents a folded structural domain; i.e., IRF-3 is a three-domain globular protein. Light scattering studies of wild-type IRF-3 showed that these three domains are tightly packed, and therefore, the dimer of IRF-3, which is formed upon phosphorylation of its C-terminal domains following virus invasion, must be a rather rigid and compact construction. One would then expect that binding of such a dimer to its tandem recognition sites PRDIII and PRDI, which are located on opposing faces of the IFN-ß enhancer DNA, should result in deformation of the DNA. Analysis of the characteristics of binding of the monomeric and dimeric IRF-3 to the enhancer DNA indeed showed that formation of this complex requires considerable work for deformation of its components, most likely bending of the DNA. Such bending was confirmed by atomic force microscopy of dimeric IRF-3 bound to the PRDII-PRDI tandem recognition sites placed at the middle of a 300 bp DNA probe. Bending of DNA by IRF-3 must be significant in the assembly and function of the IFN-ß enhancer.

Sulfated endospermic nanocellulose crystals prevent the transmission of SARS-CoV-2 and HIV-1.

Biomaterials with antimicrobial activity are gaining attention due to their biodegradability and efficacy in interacting with a wide range of microorganisms. A new cellulose nano-biomaterial, endospermic nanocellulose crystals (ENC) obtained from parenchymal tissue of ivory nut endosperm, has a natural capacity as a universal binder. This feature is enhanced when it is chemically functionalized, and can be exploited in the fight against microbes. We tested the ability of sulfated ENC in aqueous suspension to encapsulate viruses through a crosslinking reaction mediated by cations. 0.25% w/v ENC suspensions efficiently encapsulated spike (S) protein, preventing its interaction with ACE2 receptor. ENC was further able to encapsulate SARS-CoV-2 pseudoviruses and prevent infection of 293T-ACE2 cells. ENC also suppressed infection of MT-4 cells with HIV-1 LAI.04 . This antiviral activity of sulfated ENC is due to the irreversible interaction of ENC with viral particles mediated by crosslinking, as antiviral activity was less effective in the absence of cations. Additionally, ENC was used as a matrix to immobilize recombinant ACE2 receptors and anti-S IgG, creating molecular lures that efficiently inhibited SARS-CoV-2 infections in vitro . These results show that sulfated ENC from ivory nuts can be used as an efficient antiviral material.

The influence of cochlear shape on low-frequency hearing.

The conventional theory about the snail shell shape of the mammalian cochlea is that it evolved essentially and perhaps solely to conserve space inside the skull. Recently, a theory proposed that the spiral's graded curvature enhances the cochlea's mechanical response to low frequencies. This article provides a multispecies analysis of cochlear shape to test this theory and demonstrates that the ratio of the radii of curvature from the outermost and innermost turns of the cochlear spiral is a significant cochlear feature that correlates strongly with low-frequency hearing limits. The ratio, which is a measure of curvature gradient, is a reflection of the ability of cochlear curvature to focus acoustic energy at the outer wall of the cochlear canal as the wave propagates toward the apex of the cochlea.

Retinoschisin (RS1) interacts with negatively charged lipid bilayers in the presence of Ca2+: an atomic force microscopy study.

Retinoschisin (RS1) is a retina-specific secreted protein encoding a conserved discoidin domain sequence. As an adhesion molecule, RS1 preserves the retinal cell architecture and promotes visual signal transduction. In young males, loss-of-function mutations in the X-linked retinoschisis gene (RS1) cause X-linked retinoschisis, a form of progressive blindness. Neither the structure of RS1 nor the nature of its anchoring and organization on the plasma membranes is fully understood. The discoidin C2 domains of coagulation factors V and VIII are known to interact with extracellular phosphatidylserine (PS). In this study we have used atomic force microscopy (AFM) to study the interactions of murine retinoschisin (Rs1) with supported anionic lipid bilayers in the presence of Ca(2+). The bilayers consisting of a single lipid, PS, and mixtures of lipids with or without PS were used. Consistent with previous X-ray diffraction studies, AFM imaging showed two distinct domains in pure PS bilayers when Ca(2+) was present. Upon Rs1 adsorption, these PS and PS-containing mixed bilayers underwent fast and extensive reorganization. Protein localization was ascertained by immunolabeling. AFM imaging showed the Rs1 antibody bound exclusively to the calcium-rich ordered phase of the bilayers pointing to the sequestration of Rs1 within those domains. This was further supported by the increased mechanical strength of these domains after Rs1 binding. Besides, changes in bilayer thickness suggested that Rs1 was partially embedded into the bilayer. These findings support a model whereby the Rs1 protein binds to PS in the retinal cell plasma membranes in a calcium-dependent manner.

Organization of membrane motor in outer hair cells: an atomic force microscopic study.

Using atomic force microscopy, we imaged the cytosolic surface of the lateral plasma membrane of outer hair cells from guinea pigs' inner ear. We used a "cell-free" preparation, in which a patch of plasma membrane was firmly attached to a substrate and the cytoplasmic face was exposed. The membrane patches contained densely packed particles whose diameter, after correcting for the geometry of the probing tip, was approximately 10 nm. The particles were predominantly aligned unidirectionally with spacing of approximately 36 nm. The density of the particle was approximately 850 microm(-2), which could be an underestimate presumably due to the method of sample preparation. Antibody-labeled specimens showed particles more elevated than unlabeled preparation indicative of primary and secondary antibody complexes. The corrected diameters of these particles labeled with anti-actin were approximately 12 nm while that with antiprestin were approximately 8 nm. The alignment pattern in antiprestin-labeled specimens resembled that of the unlabeled preparation. Specimens labeled with actin antibodies did not show such alignment. We interpret that the particles observed in the unlabeled membranes correspond to the 10-nm particles reported by electron microscopy and that these particles contain prestin, a member of the SLC26 family, which is essential for electromotility.