Genetic code expansion in the mouse brain.

Site-specific incorporation of non-natural amino acids into proteins, via genetic code expansion with pyrrolysyl tRNA synthetase (PylRS) and tRNA(Pyl)CUA pairs (and their evolved derivatives) from Methanosarcina sp., forms the basis of powerful approaches to probe and control protein function in cells and invertebrate organisms. Here we demonstrate that adeno-associated viral delivery of these pairs enables efficient genetic code expansion in primary neuronal culture, organotypic brain slices and the brains of live mice.

Genetic code expansion enables live-cell and super-resolution imaging of site-specifically labeled cellular proteins.

Methods to site-specifically and densely label proteins in cellular ultrastructures with small, bright, and photostable fluorophores would substantially advance super-resolution imaging. Recent advances in genetic code expansion and bioorthogonal chemistry have enabled the site-specific labeling of proteins. However, the efficient incorporation of unnatural amino acids into proteins and the specific, fluorescent labeling of the intracellular ultrastructures they form for subdiffraction imaging has not been accomplished. Two challenges have limited progress in this area: (i) the low efficiency of unnatural amino acid incorporation that limits labeling density and therefore spatial resolution and (ii) the uncharacterized specificity of intracellular labeling that will define signal-to-noise, and ultimately resolution, in imaging. Here we demonstrate the efficient production of cystoskeletal proteins (ß-actin and vimentin) containing bicyclo[6.1.0]nonyne-lysine at genetically defined sites. We demonstrate their selective fluorescent labeling with respect to the proteome of living cells using tetrazine-fluorophore conjugates, creating densely labeled cytoskeletal ultrastructures. STORM imaging of these densely labeled ultrastructures reveals subdiffraction features, including nuclear actin filaments. This work enables the site-specific, live-cell, fluorescent labeling of intracellular proteins at high density for super-resolution imaging of ultrastructural features within cells.

The role of flotillins in regulating aß production, investigated using flotillin 1-/-, flotillin 2-/- double knockout mice.

Flotillin 1 and flotillin 2 associate in the plasma membrane to form microdomains that have roles in cell signaling, regulation of cell-cell contacts, membrane-cytoskeletal interactions, and endocytosis. They are thought to be involved in the trafficking and hence processing of the Amyloid Precursor Protein, APP. In this study we set out to obtain in vivo confirmation of a link between flotillins and cleavage of APP to release amyloidogenic Aß peptide, and to generate tools that would allow us to ask whether flotillins are functionally redundant. We used a mouse model for Aß-dependent cerebral amyloidosis, APPPS1 mice, combined with deletion of either flotillin 1 singly, or flotillin 1 and flotillin 2 together. There was a small but significant reduction in Aß levels, and the abundance of congo-red stained plaques, in brains of 12 week old mice lacking flotillin 1. A similar reduction in Aß levels was observed in the flotillin 1-/-, flotillin 2-/- double knockouts. We did not observe large effects on the clustering or endocytosis of APP in flotillin 1-/- mouse embryonic fibroblasts. We conclude that flotillins are likely to play some role in APP trafficking or processing, but the relevant cellular mechanisms require more investigation. The availability of flotillin 1-/-, flotillin 2-/- mice, which have no overt phenotypes, will facilitate research into flotillin function in vivo.

How do amoebae swim and crawl?

The surface behaviour of swimming amoebae was followed in cells bearing a cAR1-paGFP (cyclic AMP receptor fused to a photoactivatable-GFP) construct. Sensitized amoebae were placed in a buoyant medium where they could swim toward a chemoattractant cAMP source. paGFP, activated at the cell's front, remained fairly stationary in the cell's frame as the cell advanced; the label was not swept rearwards. Similar experiments with chemotaxing cells attached to a substratum gave the same result. Furthermore, if the region around a lateral projection near a crawling cell's front is marked, the projection and the labelled cAR1 behave differently. The label spreads by diffusion but otherwise remains stationary in the cell's frame; the lateral projection moves rearwards on the cell (remaining stationary with respect to the substrate), so that it ends up outside the labelled region. Furthermore, as cAR1-GFP cells move, they occasionally do so in a remarkably straight line; this suggests they do not need to snake to move on a substratum. Previously, we suggested that the surface membrane of a moving amoeba flows from front to rear as part of a polarised membrane trafficking cycle. This could explain how swimming amoebae are able to exert a force against the medium. Our present results indicate that, in amoebae, the suggested surface flow does not exist: this implies that they swim by shape changes.

Continuous polo-like kinase 1 activity regulates diffusion to maintain centrosome self-organization during mitosis.

Whether mitotic structures like the centrosome can self-organize from the regulated mobility of their dynamic protein components remains unclear. Here, we combine fluorescence spectroscopy and chemical genetics to study in living cells the diffusion of polo-like kinase 1 (PLK1), an enzyme critical for centrosome maturation at the onset of mitosis. The cytoplasmic diffusion of a functional EGFP-PLK1 fusion correlates inversely with known changes in its enzymatic activity during the cell cycle. Specific EGFP-PLK1 inhibition using chemical genetics enhances mobility, as do point mutations inactivating the polo-box or kinase domains responsible for substrate recognition and catalysis. Spatial mapping of EGFP-PLK1 diffusion across living cells, using raster image correlation spectroscopy and line scanning, detects regions of low mobility in centrosomes. These regions exhibit characteristics of increased transient recursive EGFP-PLK1 binding, distinct from the diffusion of stable EGFP-PLK1-containing complexes in the cytoplasm. Chemical genetic suppression of mitotic EGFP-PLK1 activity, even after centrosome maturation, causes defects in centrosome structure, which recover when activity is restored. Our findings imply that continuous PLK1 activity during mitosis maintains centrosome self-organization by a mechanism dependent on its reaction and diffusion, suggesting a model for the formation of stable mitotic structures using dynamic protein kinases.

Major translocation of calcium upon epidermal barrier insult: imaging and quantification via FLIM/Fourier vector analysis.

Calcium controls an array of key events in keratinocytes and epidermis: localized changes in Ca(2+) concentrations and their regulation are therefore especially important to assess when observing epidermal barrier homeostasis and repair, neonatal barrier establishment, in differentiation, signaling, cell adhesion, and in various pathological states. Yet, tissue- and cellular Ca(2+) concentrations in physiologic and diseased states are only partially known, and difficult to measure. Prior observations on the Ca(2+) distribution in skin were based on Ca(2+) precipitation followed by electron microscopy, or proton-induced X-ray emission. Neither cellular and/or subcellular localization could be determined through these approaches. In cells in vitro, fluorescent dyes have been used extensively for ratiometric measurements of static and dynamic Ca(2+) concentrations, also assessing organelle Ca(2+) concentrations. For lack of better methods, these findings together build the basis for the current view of the role of Ca(2+) in epidermis, their limitations notwithstanding. Here we report a method using Calcium Green 5N as the calcium sensor and the phasor-plot approach to separate raw lifetime components. Thus, fluorescence lifetime imaging (FLIM) enables us to quantitatively assess and visualize dynamic changes of Ca(2+) at light-microscopic resolution in ex vivo biopsies of unfixed epidermis, in close to in vivo conditions. Comparing undisturbed epidermis with epidermis following a barrier insult revealed major shifts, and more importantly, a mobilization of high amounts of Ca(2+) shortly following barrier disruption, from intracellular stores. These results partially contradict the conventional view, where barrier insults abrogate a Ca(2+) gradient towards the stratum granulosum. Ca(2+) FLIM overcomes prior limitations in the observation of epidermal Ca(2+) dynamics, and will allow further insights into basic epidermal physiology.

Dictyostelium amoebae and neutrophils can swim.

Animal cells migrating over a substratum crawl in amoeboid fashion; how the force against the substratum is achieved remains uncertain. We find that amoebae and neutrophils, cells traditionally used to study cell migration on a solid surface, move toward a chemotactic source while suspended in solution. They can swim and do so with speeds similar to those on a solid substrate. Based on the surprisingly rapidly changing shape of amoebae as they swim and earlier theoretical schemes for how suspended microorganisms can migrate (Purcell EM (1977) Life at low Reynolds number. Am J Phys 45:3-11), we suggest the general features these cells use to gain traction with the medium. This motion requires either the movement of the cell's surface from the cell's front toward its rear or protrusions that move down the length of the elongated cell. Our results indicate that a solid substratum is not a prerequisite for these cells to produce a forward thrust during movement and suggest that crawling and swimming are similar processes, a comparison we think is helpful in understanding how cells migrate.

Multimodal CARS microscopy determination of the impact of diet on macrophage infiltration and lipid accumulation on plaque formation in ApoE-deficient mice.

We characterized several cellular and structural features of early stage Type II/III atherosclerotic plaques in an established model of atherosclerosis-the ApoE-deficient mouse-by using a multimodal, coregistered imaging system that integrates three nonlinear optical microscopy (NLOM) contrast mechanisms: coherent anti-Stokes Raman scattering (CARS), second harmonic generation (SHG), and two-photon excitation fluorescence (TPEF). Specifically, the infiltration of lipid-rich macrophages and the structural organization of collagen and elastin fibers were visualized by CARS, SHG, and TPEF, respectively, in thick tissue specimens without the use of exogenous labels or dyes. Label-free CARS imaging of macrophage accumulation was confirmed by histopathology using CD68 staining. A high-fat, high-cholesterol Western diet resulted in an approximate 2-fold increase in intimal plaque area, defined by CARS signals of lipid-rich macrophages. Additionally, analysis of collagen distribution within lipid-rich plaque regions revealed nearly a 4-fold decrease in the Western diet-fed mice, suggesting NLOM sensitivity to increased matrix metalloproteinase (MMP) activity and decreased smooth muscle cell (SMC) accumulation. These imaging results provide significant insight into the structure and composition of early stage Type II/III plaque during formation and allow for quantitative measurements of the impact of diet and other factors on critical plaque and arterial wall features.

Liver cyst cytokines promote endothelial cell proliferation and development.

Autosomal dominant polycystic kidney (ADPKD) is highly prevalent genetic disease. Liver cyst disease is the most common extrarenal manifestation in ADPKD and accounts for up to 10% of ADPKD morbidity and mortality. The clinical features of ADPKD liver disease arise from dramatic increases in liver cyst volumes. To identify mechanisms that promote liver cyst growth, the present study characterized the degree of vascularization of liver cyst walls and determined that cyst-specific cytokines and growth factors can drive endothelial cell proliferation and development. Microscopic techniques demonstrated liver cyst walls are well vascularized. A comparative analysis found the vascular density in free liver cyst walls was greater in mice than in humans. Treatment of human micro-vascular endothelial cells (HMEC-1) with human liver cyst fluid (huLCF) induced a rapid increase in vascular endothelium growth factor receptor 2 (VEGFR2) phosphorylation that persisted for 45-60 min and was blocked by 20 microM SU5416, a VEGFR tyrosine kinase inhibitor. Similarly, huLCF treatment of HMEC-1 cells induced an increase in the cell proliferation rate (131 +/- 6% of control levels; P > 0.05) and the degree of vascular development ('tube' diameter assay: 92 +/- 14 microm for huLCF vs. 12 +/- 7 microm for vehicle); P > 0.05). Both cell proliferation and vascular development were sensitive to SU5416. These studies indicate that factors secreted by liver cyst epithelia can activate VEGF signaling pathways and induce endothelial cell proliferation and differentiation. The present studies suggest that targeting VEGFR2-dependent angiogenesis may be an effective therapeutic strategy in blocking ADPKD liver cyst vascularization and growth.

Differential regulation of the renal sodium-phosphate cotransporters NaPi-IIa, NaPi-IIc, and PiT-2 in dietary potassium deficiency.

Dietary potassium (K) deficiency is accompanied by phosphaturia and decreased renal brush border membrane (BBM) vesicle sodium (Na)-dependent phosphate (P(i)) transport activity. Our laboratory previously showed that K deficiency in rats leads to increased abundance in the proximal tubule BBM of the apical Na-P(i) cotransporter NaPi-IIa, but that the activity, diffusion, and clustering of NaPi-IIa could be modulated by the altered lipid composition of the K-deficient BBM (Zajicek HK, Wang H, Puttaparthi K, Halaihel N, Markovich D, Shayman J, Beliveau R, Wilson P, Rogers T, Levi M. Kidney Int 60: 694-704, 2001; Inoue M, Digman MA, Cheng M, Breusegem SY, Halaihel N, Sorribas V, Mantulin WW, Gratton E, Barry NP, Levi M. J Biol Chem 279: 49160-49171, 2004). Here we investigated the role of the renal Na-P(i) cotransporters NaPi-IIc and PiT-2 in K deficiency. Using Western blotting, immunofluorescence, and quantitative real-time PCR, we found that, in rats and in mice, K deficiency is associated with a dramatic decrease in the NaPi-IIc protein abundance in proximal tubular BBM and in NaPi-IIc mRNA. In addition, we documented the presence of a third Na-coupled P(i) transporter in the renal BBM, PiT-2, whose abundance is also decreased by dietary K deficiency in rats and in mice. Finally, electron microscopy showed subcellular redistribution of NaPi-IIc in K deficiency: in control rats, NaPi-IIc immunolabel was primarily in BBM microvilli, whereas, in K-deficient rats, NaPi-IIc BBM label was reduced, and immunolabel was prevalent in cytoplasmic vesicles. In summary, our results demonstrate that decreases in BBM abundance of the phosphate transporter NaPi-IIc and also PiT-2 might contribute to the phosphaturia of dietary K deficiency, and that the three renal BBM phosphate transporters characterized so far can be differentially regulated by dietary perturbations.

Interaction of MAP17 with NHERF3/4 induces translocation of the renal Na/Pi IIa transporter to the trans-Golgi.

The function of the NaPiIIa renal sodium-phosphate transporter is regulated through a complex network of interacting proteins. Several PDZ domain-containing proteins interact with its COOH terminus while the small membrane protein MAP17 interacts with its NH(2) end. To elucidate the function of MAP17, we identified its interacting proteins using both bacterial and mammalian two-hybrid systems. Several PDZ domain-containing proteins, including the four NHERF proteins, as well as NaPiIIa and NHE3, were found to bind to MAP17. The interactions of MAP17 with the NHERF proteins and with NaPiIIa were further analyzed in opossum kidney (OK) cells. Expression of MAP17 alone had no effect on the NaPiIIa apical membrane distribution, but coexpression of MAP17 and NHERF3 or NHERF4 induced internalization of NaPiIIa, MAP17, and the PDZ protein to the trans-Golgi network (TGN). This effect was not observed when MAP17 was cotransfected with NHERF1/2 proteins. Inhibition of protein kinase C (PKC) prevented expression of the three proteins in the TGN. Activation of PKC in OK cells transfected only with MAP17 induced complete degradation of MAP17 and NaPiIIa. When lysosomal degradation was prevented, both proteins accumulated in the TGN. When the dopamine D1-like receptor was activated with fenoldopam, both NaPiIIa and MAP17 also accumulated in the TGN. Finally, cotransfection of MAP17 and NHERF3 prevented the adaptive upregulation of phosphate transport activity in OK cells in response to low extracellular phosphate. Therefore, the interaction between MAP17, NHERF3/4, and NaPiIIa in the TGN could be an important intermediate or alternate path in the internalization of NaPiIIa.

Fluorescence correlation spectroscopy and fluorescence lifetime imaging microscopy.

With few and commercially available add-ons, both confocal and full-field fluorescence microscopes can be adapted to provide more information on the biological sample of interest. In this review we discuss the possibilities offered by two additional functionalities to fluorescence microscopes, fluorescence correlation spectroscopy (FCS) and fluorescence lifetime imaging mi croscopy (FLIM). FCS measurements at a single point in a sample allow kinetic and diffusion properties of fluorescently labeled molecules to be determined, as well as their concentration and aggregation state. Data from multiple points of the sample can be acquired using scanning-FCS, image correlation spectroscopy, and raster image correlation spectroscopy. These techniques cover phenomena with characteristic durations from sub-microsecond to second time scales. The power of FLIM lies in the fact that the measured fluorescent lifetime of a fluorophore is sensitive to the molecular environment of that fluorophore. FLIM is a robust means to quantify Forster resonance energy transfer and thus determine protein-protein interactions or protein conformational changes. In addition, FLIM is very valuable for functional imaging of ion concentrations in cells and tissues as it can be applied in heterogeneously labeled samples. In summary, FCS and FLIM allow information to be gathered beyond localization, including diffusional mobility, protein clustering and interactions, and molecular environment.

Altered renal tubular expression of the complement inhibitor Crry permits complement activation after ischemia/reperfusion.

Ischemia/reperfusion (I/R) of several organs results in complement activation, but the kidney is unique in that activation after I/R occurs only via the alternative pathway. We hypothesized that selective activation of this pathway after renal I/R could occur either because of a loss of complement inhibition or from increased local synthesis of complement factors. We examined the relationship between renal complement activation after I/R and the levels and localization of intrinsic membrane complement inhibitors. We found that loss of polarity of complement receptor 1-related protein y (Crry) in the tubular epithelium preceded activation of the alternative pathway along the basolateral aspect of the tubular cells. Heterozygous gene-targeted mice that expressed lower amounts of Crry were more sensitive to ischemic injury. Furthermore, inhibition of Crry expressed by proximal tubular epithelial cells in vitro resulted in alternative pathway-mediated injury to the cells. Thus, altered expression of a complement inhibitor within the tubular epithelium appears to be a critical factor permitting activation of the alternative pathway of complement after I/R. Increased C3 mRNA and decreased factor H mRNA were also detected in the outer medulla after I/R, suggesting that altered synthesis of these factors might further contribute to complement activation in this location.

Acute and chronic changes in cholesterol modulate Na-Pi cotransport activity in OK cells.

We previously showed an inverse correlation between membrane cholesterol content and Na-P(i) cotransport activity during the aging process and adaptation to alterations in dietary P(i) in the rat (Levi M, Jameson DM, and van der Meer BW. Am J Physiol Renal Fluid Electrolyte Physiol 256: F85-F94, 1989). The purpose of the present study was to determine whether alterations in cholesterol content per se modulate Na-P(i) cotransport activity and apical membrane Na-P(i) protein expression in opossum kidney (OK) cells. Acute cholesterol depletion achieved with beta-methyl cyclodextrin (beta-MCD) resulted in a significant increase in Na-P(i) cotransport activity accompanied by a moderate increase in apical membrane Na-P(i) protein abundance and no alteration of total cellular Na-P(i) protein abundance. Conversely, acute cholesterol enrichment achieved with beta-MCD/cholesterol resulted in a significant decrease in Na-P(i) cotransport activity with a moderate decrease in apical membrane Na-Pi protein abundance and no change of the total cellular Na-P(i) protein abundance. In contrast, chronic cholesterol depletion, achieved by growing cells in lipoprotein-deficient serum (LPDS), resulted in parallel and significant increases in Na-P(i) cotransport activity and apical membrane and total cellular Na-P(i) protein abundance. Cholesterol depletion also resulted in a significant increase in membrane lipid fluidity and alterations in lipid microdomains as determined by laurdan fluorescence spectroscopy and imaging. Chronic cholesterol enrichment, achieved by growing cells in LPDS followed by loading with low-density lipoprotein, resulted in parallel and significant decreases in Na-P(i) cotransport activity and apical membrane and total cellular Na-P(i) protein abundance. Our results indicate that in OK cells acute and chronic alterations in cholesterol content per se modulate Na-P(i) cotransport activity by diverse mechanisms that also include significant interactions of Na-P(i) protein with lipid microdomains.

Partitioning of NaPi cotransporter in cholesterol-, sphingomyelin-, and glycosphingolipid-enriched membrane domains modulates NaPi protein diffusion, clustering, and activity.

In dietary potassium deficiency there is a decrease in the transport activity of the type IIa sodium/phosphate cotransporter protein (NaPi) despite an increase in its apical membrane abundance. This novel posttranslational regulation of NaPi activity is mediated by the increased glycosphingolipid content of the potassium-deficient apical membrane. However, the mechanisms by which these lipids modulate NaPi activity have not been determined. We determined if in potassium deficiency NaPi is increasingly partitioned in cholesterol-, sphingomyelin-, and glycosphingolipid-enriched microdomains of the apical membrane and if the increased presence of NaPi in these microdomains modulates its activity. By using a detergent-free density gradient flotation technique, we found that 80% of the apical membrane NaPi partitions into the low density cholesterol-, sphingomyelin-, and GM1-enriched fractions characterized as "lipid raft" fractions. In potassium deficiency, a higher proportion of NaPi was localized in the lipid raft fractions. By combining fluorescence correlation spectroscopy and photon counting histogram methods for control and potassium-deficient apical membranes reconstituted into giant unilamellar vesicles, we showed a 2-fold decrease in lateral diffusion of NaPi protein and a greater than 2-fold increase in size of protein aggregates/clusters in potassium deficiency. Our results indicate that NaPi protein is localized in membrane microdomains, that in potassium deficiency a larger proportion of NaPi protein is present in these microdomains, and that NaPi lateral diffusion is slowed down and NaPi aggregation/clustering is increased in potassium deficiency, both of which could be associated with the decreased Na/Pi cotransport activity in potassium deficiency.

Neonatal development of the stratum corneum pH gradient: localization and mechanisms leading to emergence of optimal barrier function.

Although basal permeability barrier function is established at birth, the higher risk for infections, dermatitis, and percutaneous absorption of toxic agents may indicate incomplete permeability barrier maturation in the early neonatal period. Since stratum corneum (SC) acidification in adults is required for normal permeability barrier homeostasis, and lipid processing occurs via acidic pH dependent enzymes, we hypothesized that, in parallel with the less acidic surface pH, newborn SC would exhibit signs of incomplete barrier formation. Fluorescence lifetime imaging reveals that neonatal rat SC acidification first becomes evident by postnatal day 3, in extracellular "microdomains" at the SC- stratum granulosum (SG) interface, where pH-sensitive lipid processing is known to occur. This localized acidification correlated temporally with efficient processing of secreted lamellar body contents to mature extracellular lamellar bilayers. Since expression of the key acidifying mechanism NHE1 is maximal just prior to birth, and gradually declines over the first postnatal week, suboptimal SC acidification at birth cannot be attributed to insufficient NHE1 expression, but could instead reflect reduced NHE1 activity. Expression of the key lipid processing enzyme, beta-glucocerebrosidase (beta-GlcCer'ase), develops similar to NHE1, excluding a lack of beta-GlcCer'ase protein as rate limiting for efficient lipid processing. These results define a postnatal development consisting of initial acidification in the lower SC followed by outward progression, which is accompanied by formation of mature extracellular lamellar membranes. Thus, full barrier competence appears to require the extension of acidification in microdomains from the SC/SG interface outward toward the skin surface in the immediate postnatal period.

NHE1 regulates the stratum corneum permeability barrier homeostasis. Microenvironment acidification assessed with fluorescence lifetime imaging.

The outermost epidermal layer, the stratum corneum (SC), exhibits an acidic surface pH, whereas the pH at its base approaches neutrality. NHE1 is the only Na(+)/H(+) antiporter isoform in keratinocytes and epidermis, and has been shown to regulate intracellular pH. We now demonstrate a novel function for NHE1, as we find that it also controls acidification of extracellular "microdomains" in the SC that are essential for activation of pH-sensitive enzymes and the formation of the epidermal permeability barrier. NHE1 expression in epidermis is most pronounced in granular cell layers, and although the surface pH of NHE1 knockout mice is only slightly more alkaline than normal using conventional pH measurements, a more sensitive method, fluorescence lifetime imaging, demonstrates that the acidic intercellular domains at the surface and of the lower SC disappear in NHE1 -/- animals. Fluorescence lifetime imaging studies also reveal that SC acidification does not occur through a uniform gradient, but through the progressive accumulation of acidic microdomains. These findings not only visualize the spatial distribution of the SC pH gradient, but also demonstrate a role for NHE1 in the generation of acidic extracellular domains of the lower SC, thus providing the acidification of deep SC interstices necessary for lipid processing and barrier homeostasis.

Two-photon fluorescence lifetime imaging of the skin stratum corneum pH gradient.

Two-photon fluorescence lifetime imaging is used to identify microdomains (1-25 microm) of two distinct pH values within the uppermost layer of the epidermis (stratum corneum). The fluorophore used is 2',7'-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein (BCECF), whose lifetime tau (pH 4.5, tau = 2.75 ns; pH 8.5, tau = 3.90 ns) is pH dependent over the pH range of the stratum corneum (pH 4.5 to pH 7.2). Hairless mice (SKH1-hrBR) are used as a model for human skin. Images (< or =50 microm x 50 microm) are acquired every 1.7 microm from the stratum corneum surface to the first viable layer (stratum granulosum). Acidic microdomains (average pH 6.0) of variable size (~1 microm in diameter with variable length) are detected within the extracellular matrix of the stratum corneum, whereas the intracellular space of the corneocytes in mid-stratum corneum (25 microm diameter) approaches neutrality (average pH 7.0). The surface is acidic. The average pH of the stratum corneum increases with depth because of a decrease in the ratio of acidic to neutral regions within the stratum corneum. The data definitively show that the stratum corneum acid mantle results from the presence of aqueous acidic pockets within the lipid-rich extracellular matrix.