Architecture of kangaroo rat inner medulla: segmentation of descending thin limb of Henle's loop

We hypothesize that the inner medulla of the kangaroo rat Dipodomys merriami, a desert rodent that concentrates its urine to more than 6,000 mosmol/kgH(2)O water, provides unique examples of architectural features necessary for production of highly concentrated urine. To investigate this architecture, inner medullary nephron segments in the initial 3,000 μm below the outer medulla were assessed with digital reconstructions from physical tissue sections. Descending thin limbs of Henle (DTLs), ascending thin limbs of Henle (ATLs), and collecting ducts (CDs) were identified by immunofluorescence using antibodies that label segment-specific proteins associated with transepithelial water flux (aquaporin 1 and 2, AQP1 and AQP2) and chloride flux (the chloride channel ClC-K1); all tubules and vessels were labeled with wheat germ agglutinin. In the outer 3,000 μm of the inner medulla, AQP1-positive DTLs lie at the periphery of groups of CDs. ATLs lie inside and outside the groups of CDs. Immunohistochemistry and reconstructions of loops that form their bends in the outer 3,000 μm of the inner medulla show that, relative to loop length, the AQP1-positive segment of the kangaroo rat is significantly longer than that of the Munich-Wistar rat. The length of ClC-K1 expression in the prebend region at the terminal end of the descending side of the loop in kangaroo rat is about 50% shorter than that of the Munich-Wistar rat. Tubular fluid of the kangaroo rat DTL may approach osmotic equilibrium with interstitial fluid by water reabsorption along a relatively longer tubule length, compared with Munich-Wistar rat. A relatively shorter-length prebend segment may promote a steeper reabsorptive driving force at the loop bend. These structural features predict functionality that is potentially significant in the production of a high urine osmolality in the kangaroo rat.     

Ultrastructural localization of Na+,K+-ATPase in rat and rabbit kidney medulla

Na+,K+-ATPase was localized at the ultrastructural level in rat and rabbit kidney medulla. The cytochemical method for the K+-dependent phosphatase component of the enzyme, using p-nitrophenylphosphate (NPP) as substrate, was employed to demonstrate the distribution of Na+, K+- ATPase in tissue-chopped sections from kidneys perfusion-fixed with 1% paraformaldehyde-0.25% glutaraldehyde. In other outer medulla of rat kidney, ascending thick limbs (MATL) were sites of intense K+-dependent NPPase (K+-NPPase) activity, whereas descending thick limbs and collecting tubules were barely reactive. Although descending thin limbs (DTL) of short loop nephrons were unstained, DTL from long loop nephrons in outer medulla were sites of moderate K+-NPPase activity. In rat inner medulla, DTL and ascending thin limbs (ATL) were unreactive for K+-NPPase. In rabbit medulla, only MATL were sites of significant K+-NPPase activity. The specificity of the cytochemical localization of Na+,K+-ATPase at reactive sites in rat and rabbit kidney medulla was demonstrated by K+-dependence of reaction product deposition, localization of reaction product (precipitated phosphate hydrolyzed from NPP) to the cytoplasmic side of basolateral plasma membranes, insensitivity of the reaction to inhibitors of nonspecific alkaline phosphatase, and, in the glycoside-sensitive rabbit kidney, substantial inhibition of staining by ouabain. The observed pattern of distribution of the sodium transport enzyme in kidney medulla is particularly relevant to current models for urine concentration. The presence of substantial Na+,K+-ATPase in MATL is consistent with the putative role of this segment as the driving force for the countercurrent multiplication system in the outer medulla. The absence of significant activity in inner medullary ATL and DTL, however, implies that interstitial solute accumulation in this region probably occurs by passive processes. The localization of significant Na+,K+-ATPase in outer medullary DTL of long loop nephrons in the rat suggests that solute addition in this segment may occur in part by an active salt secretory mechanism that could ultimately contribute to the generation of inner medullary interstitial hypertonicity and urine concentration.     

Properties of soluble cyclic AMP-dependent protein kinase activity of renal inner medulla

Studies of the chromatographic distribution of soluble protein kinase in rat kidney demonstrated that the type I isoenzyme predominates in cortex, whereas activity in outer and inner medulla is almost exclusively the type II form. The type II isoenzyme also predominates (95% or greater) in human, canine, bovine, porcine and rabbit inner medulla. Compared to soluble type I activities from rat renal cortex or medulla, type II activity of inner medulla demonstrates a marked resistance to activation by NaCl and/or urea in subcellular preparations. However, with respect to solute activation, the resistance of the type II enzyme of inner medulla does not differ from that of type II activities from other tissues. In contrast to the effects on basal activity, NaCl and urea potentiated inner medullary type II activation by cyclic AMP and also delayed the rate of subunit reassociation after chromatographic removal of cyclic AMP. Incubation of inner medullary slices in high osmolality buffer (NaCl and urea) did not alone activate soluble protein kinase, an observation which implied that the enzyme was also resistant to solute activation in the intact cell system. Moreover, at 1650 mosM, vasopressin activation of soluble protein kinase was enhanced compared to responses at 750 mosM despite comparabel levels of cyclic AMP accumulation at the two osmolalities. However, a cyclic AMP-independent action of high osmolality to reduce the rate of inactivation of arginine vasopressin-stimulated protein kinase was not demonstrable in inner medullary slices.The present data suggest the possibility that the resistance of inner medullary protein kinase to solute activation could be related to the isomeric form of enzyme (type II) present in this tissue. The high concentrations of NaCl and urea routinely found in inner medulla during hydropenia also influenced protein kinase responses to arginine vasopressin, and may do so in part by directly potentiating the action of cyclic AMP on subunit dissociation.     

Countercurrent exchange in the renal medulla

The microcirculation of the renal medulla traps NaCl and urea deposited to the interstitium by the loops of Henle and collecting ducts. Theories have predicted that countercurrent exchanger efficiency is favored by high permeability to solute. In contrast to the conceptualization of vasa recta as simple "U-tube" diffusive exchangers, many findings have revealed surprising complexity. Tubular-vascular relationships in the outer and inner medulla differ markedly. The wall structure and transport properties of descending vasa recta (DVR) and ascending vasa recta (AVR) are very different. The recent discoveries of aquaporin-1 (AQP1) water channels and the facilitated urea carrier UTB in DVR endothelia show that transcellular as well as paracellular pathways are involved in equilibration of DVR plasma with the interstitium. Efflux of water across AQP1 excludes NaCl and urea, leading to the conclusion that both water abstraction and diffusion contribute to transmural equilibration. Recent theory predicts that loss of water from DVR to the interstitium favors optimization of urinary concentration by shunting water to AVR, secondarily lowering blood flow to the inner medulla. Finally, DVR are vasoactive, arteriolar microvessels that are anatomically positioned to regulate total and regional blood flow to the outer and inner medulla. In this review, we provide historical perspective, describe the current state of knowledge, and suggest areas that are in need of further exploration.     

Concentrating Engines and the Kidney: II. Multisolute Central Core Systems

The analysis of the central core model of the renal medulla is extended to multisolute systems. It is shown that total solute concentration obeys the same differential equations for core and ascending limb as in a single solute system. Equations are derived for the concentration of individual solutes. Application of these equations to a two solute system shows that a central core system can concentrate with all transport being down a concentration gradient. This analysis applied to the renal medulla shows that mixing of urea from the collecting duct (CD) and salt from the loop of Henle in the central core of the inner medulla contributes to the concentration of urine during antidiuresis. It also sets criteria for completely passive function of the loop in the inner medulla, but whether these are satisfied cannot be determined from present experimental data.     

Maximum Urine Concentrating Capability in a Mathematical Model of the Inner Medulla of the Rat Kidney

In a mathematical model of the urine concentrating mechanism of the inner medulla of the rat kidney, a nonlinear optimization technique was used to estimate parameter sets that maximize the urine-to-plasma osmolality ratio (U/P) while maintaining the urine flow rate within a plausible physiologic range. The model, which used a central core formulation, represented loops of Henle turning at all levels of the inner medulla and a composite collecting duct (CD). The parameters varied were: water flow and urea concentration in tubular fluid entering the descending thin limbs and the composite CD at the outer-inner medullary boundary; scaling factors for the number of loops of Henle and CDs as a function of medullary depth; location and increase rate of the urea permeability profile along the CD; and a scaling factor for the maximum rate of NaCl transport from the CD. The optimization algorithm sought to maximize a quantity E that equaled U/P minus a penalty function for insufficient urine flow. Maxima of E were sought by changing parameter values in the direction in parameter space in which E increased. The algorithm attained a maximum E that increased urine osmolality and inner medullary concentrating capability by 37.5% and 80.2%, respectively, above base-case values; the corresponding urine flow rate and the concentrations of NaCl and urea were all within or near reported experimental ranges. Our results predict that urine osmolality is particularly sensitive to three parameters: the urea concentration in tubular fluid entering the CD at the outer-inner medullary boundary, the location and increase rate of the urea permeability profile along the CD, and the rate of decrease of the CD population (and thus of CD surface area) along the cortico-medullary axis.     

Intrarenal distribution of calcium and magnesium in the dog. Effect of an osmotic diuresis (author's transl)]

1 The determination of Na, Ca, Mg, and K concentrations was performed in four different regions of the dog kidney (cortex, outer medulla, inner medulla, and papilla) during antidiuresis and during an osmotic diuresis. 2 The results show a medullary concentration gradient for calcium. This gradient is much higher than that found for sodium. 3 An inverse concentration gradient from cortex to inner medulla for Mg and K is found. 4 An osmotic diuresis (hypertonic mannitol) decreases the corticomedullary gradient of Na, but does not alter significantly the intrarenal distribution of Ca, Mg and K. 5 These results consistent with an intracellular localization of Mg and K in the renal tissue. It is suggested that the medullary concentration gradient for Ca may be due either to a countercurrent multiplier system similar to that for Na, or to a higher tissular fixation of Ca in the inner medulla and papilla than in the outer medulla and cortex.     

Renal medullary electrolytes: effects of furosemide assessed by studies in vivo of electrical admittance

In anaesthetized rabbits electrical admittance (a reciprocal of impedance) of the kidney in situ was recorded using electrodes located in the cortex, outer medulla, inner medulla and papilla. Renal haemodynamics, clearances and Na+ concentration in tissue slices were also determined. Admittance changes in response to i.v. furosemide, 1.5 or 3 mg/kg body weight, and to 15% mannitol infusion, reflected changing interstitial electrolyte concentration and, indirectly, changes in tubular reabsorption of NaCl. The large dose of furosemide and mannitol infusion decreased admittance in all renal zones whereas the small dose affected only the inner medulla and papilla. The rapid onset of the fall in admittance of the inner medulla, even in absence of changes within the outer medulla, suggests that the drug's action is not confined to the thick ascending limb but includes the thin ascending segment.     

Concentrating engines and the kidney. III. Canonical mass balance equation for multinephron models of the renal medulla.

The canonical mass balance relation derived for the central core model of the renal medulla is extended to medullary models in which an arbitrary assemblage of renal tubules and vascular capillaries exchange with each other both directly and via the medullary interstitium and in which not all of the vascular loops or loops of Henle extend to the papilla. It is shown that if descending limbs of Henle and descending vasa recta enter the medulla at approximately plasma osmolality, the concentration ratio is given by: r = 1/[1 - ft(1 - fu)(1 - fw)], where ft is fractional solute transport out of ascending Henle's limb, fu is fractional urine flow, and fw is fractional dissipation; fw is a measure of the solute returned to the systemic circulation without its isotonic complement of water. A modified equation that applies to the diluting as well as the concentrating kidney is also derived. By allowing concentrations in interstitium and vascular capillaries to become identical at a given medullary level, conservation relations are derived for a multinephron central core model of the renal medulla.     

Basolateral glucose transport in distal segments of the dog nephron

The transport of glucose by canine thick ascending limbs (TAL) and inner medullary collecting ducts (IMCD) was studied using tubule suspensions and membrane vesicles. The uptake of D-[14C(U)]glucose by a suspension of intact TAL tubules was reduced largely by phloretin (Pt), moderately by phlorizin (Pz), and completely suppressed by a combination of both agents. A selective effect of Pz on the transport of [14C]alpha-methyl-D-glucoside, but not on 2-[3H]deoxyglucose, was also observed in TAL tubules. In contrast, glucose transport was unaffected by Pz but entirely suppressed by Pt alone in IMCD tubules. The metabolism of glucose was largely suppressed by Pt but unaffected by Pz in both types of tubules. Membrane vesicles were prepared from the red medulla and the white papilla or from TAL and IMCD tubules isolated from these tissues. Vesicle preparations from both tissues demonstrated a predominant carrier-mediated, sodium-independent, Pt- and cytochalasin B-sensitive glucose transport. Following purification of basolateral membrane on a Percoll gradient, the sodium-insensitive D-[14C(U)]glucose transport activity copurified with the activity of the basolateral marker Na(+)-K+ ATPase in both tissues. However, a small sodium-dependent and Pz-sensitive component of glucose transport was found in membrane vesicles prepared from the red medulla or from thick ascending limb tubules but not from the papilla nor collecting duct tubules. The kinetic analysis of the major sodium-independent processes showed that the affinity of the transporter for glucose was greater in collecting ducts (Km = 2.3 mM) than in thick ascending limbs (Km = 4.9 mM). We conclude that glucose gains access into the cells largely through a basolateral facilitated diffusion process in both segments. However a small sodium-glucose cotransport is also detected in membranes of TAL tubules. The transport of glucose presents an axial differentiation in the affinity of glucose transporters in the renal medulla, ensuring an adequate supply of glucose to the glycolytic inner medullary structures.     

Lipid microdomain clustering induces a redistribution of antigen recognition and adhesion molecules on human T lymphocytes

The study of lipid microdomains in the plasma membrane is a topic of recent interest in leukocyte biology. Many T cell activation and signaling molecules are found to be associated with lipid microdomains and have been implicated in normal T cell function. It has been proposed that lipid microdomains with their associated molecules move by lateral diffusion to areas of cellular interactions to initiate signaling pathways. Using sucrose density gradients we have found that human T cell beta(1) integrins are not normally associated with lipid microdomains. However, cross-linking of GM1 through cholera toxin B-subunit (CTB) causes an enrichment of beta(1) integrins in microdomain fractions, suggesting that cross-linking lipid microdomains causes a reorganization of molecular associations. Fluorescent microscopy was used to examine the localization of various lymphocyte surface molecules before and after lipid microdomain cross-linking. Lymphocytes treated with FITC-CTB reveal an endocytic vesicle that is enriched in TCR and CD59, while beta(1) integrin, CD43, and LFA-3 were not localized in the vesicle. However, when anti-CTB Abs are used to cross-link lipid microdomains, the microdomains are not internalized but are clustered on the cell surface. In this study, CD59, CD43, and beta(1) integrin are all seen to colocalize in a new lipid microdomain from which LFA-3 remains excluded and the TCR is now dissociated. These findings show that cross-linking lipid microdomains can cause a dynamic rearrangement of the normal order of T lymphocyte microdomains into an organization where novel associations are created and signaling pathways may be initiated.     

Polyol determination along the rat nephron

The polyols sorbitol and inositol were determined in single freshly microdissected tubule segments of rat kidney. Twenty different structures were separated from six different kidney zones reaching from cortex to papillary tip. Picomol amounts of sorbitol and inositol were quantitated by use of an enzymatic bioluminescence procedure. Experimental conditions (700 mosmol/kg, 4 degrees C) were chosen to assure constant polyol concentrations over 3 h dissection period. Sorbitol exhibited a concentration gradient in the collecting duct system from the outer/inner medullary border (3.9 +/- 0.5 pmol/mm) to the papillary tip (78.8 +/- 6.9 pmol/mm). In the same region descending and ascending limbs of Henle's loop contained 1.5 +/- 0.5 to 5.3 +/- 1.6 pmol/mm and 2.5 +/- 0.8 to 8.35 +/- 1.5 pmol/mm, respectively. In contrast, all outer medullary and cortical structures had lower sorbitol concentrations. Inositol amounts increased continuously in the collecting duct from cortex (5.3 +/- 0.5 pmol/mm) to inner medulla (30.7 +/- 3.8 pmol/mm). This polyol was also found in thick ascending limb of Henle's loop (6.2 +/- 1.1 pmol/mm in cortex to 11.2 +/- 1.4 pmol/mm in outer medulla) and in proximal tubules (5.6 +/- 1.2 pmol/mm in S1 and 4.5 +/- 1.5 pmol/mm in S3). When related to cellular volume measured by planimetry, intracellular sorbitol concentration was calculated to be 51 mmol/l in papillary collecting duct and inositol 28 mmol/l in outer medullary thick ascending limb cells. These data confirm the role of sorbitol in the renal concentrating process in papilla. Inositol seems to have additional function in thick ascending limb of Henle's loop and the proximal tubule.     

An inverse algorithm for a mathematical model of an avian urine concentrating mechanism

A nonlinear optimization technique, in conjunction with a single-nephron, single-solute mathematical model of the quail urine concentrating mechanism, was used to estimate parameter sets that optimize a measure of concentrating mechanism efficiency, viz., the ratio of the free-water absorption rate to the total NaCl active transport rate. The optimization algorithm, which is independent of the numerical method used to solve the model equations, runs in a few minutes on a 1000 MHz desktop computer. The parameters varied were: tubular permeabilities to water and solute; maximum active solute transport rates of the ascending limb of Henle and the collecting duct (CD); length of the prebend enlargement (PBE) of the descending limb; fractional solute delivery to the CD; solute concentration of tubular fluid entering the CD at the cortico-medullary boundary; and rate of exponential CD population decrease along the medullary cone. Using a base-case parameter set and parameter bounds suggested by physiologic experiments, the optimization algorithm identified a maximum-efficiency set of parameter values that increased efficiency by 40% above base-case efficiency; a minimum-efficiency set reduced efficiency by about 41%. When maximum-efficiency parameter values were computed as medullary length varied over the physiologic range, the PBE was found to make up 88% of a short medullary cone but only 8% of a long medullary cone.     

Developmental immunolocalization of heat shock protein 70 (HSP70) in epithelial cell of rat kidney

During renal development the cells in the medulla are exposed to elevated and variable interstitial osmolality. Heat shock protein 70 (HSP70) is a major molecular chaperone and plays an important role in the protection of cells in the renal medulla from high osmolality. The purpose of this study was to establish the time of immunolocalization and distribution of HSP70 in developing and adult rat kidney. In addition, changes in HSP70 immunolocalization following the infusion of furosemide were investigated. In adult animals, the HSP70 was expressed in the medullary thin ascending limb of Henle's loop (ATL) and inner medullary collecting duct (IMCD). In developing kidney, HSP70 immunoreactivity was first detected in the IMCD of the papillary tip on postnatal day 1. From four to 14 days of age, HSP70 was detected in the ATL after transformation from thick ascending limb, beginning at the papillary tip and ascending to the border between the outer and inner medulla. The immunolocalization of HSP70 in both the ATL and IMCD gradually increased during two weeks. The gradual increase in HSP70 was associated with an increase in its mRNA abundance. However, furosemide infusion resulted in significantly reduced HSP70 immunolocalization in the IMCD and ATL. These data demonstrated that the expression of HSP70 was closely correlated with changes in interstitial osmolality during the development of the kidney. We suggest that HSP70 protects ATL and IMCD cells in the inner medulla from the stress of high osmolality and may be involved in the transformation of the ATL of the long loop of Henle during renal development.     

A mathematical model of the urine concentrating mechanism in the rat renal medulla. II. Functional implications of three-dimensional architecture

In a companion study [Layton AT. A mathematical model of the urine concentrating mechanism in the rat renal medulla. I. Formulation and base-case results. Am J Physiol Renal Physiol. (First published November 10, 2010). 10.1152/ajprenal.00203.2010] a region-based mathematical model was formulated for the urine concentrating mechanism in the renal medulla of the rat kidney. In the present study, we investigated model sensitivity to some of the fundamental structural assumptions. An unexpected finding is that the concentrating capability of this region-based model falls short of the capability of models that have radially homogeneous interstitial fluid at each level of only the inner medulla (IM) or of both the outer medulla and IM, but are otherwise analogous to the region-based model. Nonetheless, model results reveal the functional significance of several aspects of tubular segmentation and heterogeneity: 1) the exclusion of ascending thin limbs that reach into the deep IM from the collecting duct clusters in the upper IM promotes urea cycling within the IM; 2) the high urea permeability of the lower IM thin limb segments allows their tubular fluid urea content to equilibrate with the surrounding interstitium; 3) the aquaporin-1-null terminal descending limb segments prevent water entry and maintain the transepithelial NaCl concentration gradient; 4) a higher thick ascending limb Na(+) active transport rate in the inner stripe augments concentrating capability without a corresponding increase in energy expenditure for transport; 5) active Na(+) reabsorption from the collecting duct elevates its tubular fluid urea concentration. Model calculations predict that these aspects of tubular segmentation and heterogeneity promote effective urine concentrating functions.     

Effects of carotid baroreceptor stimulation and renal denervation on the medullary concentration gradient in rat kidneys.

Unilateral stimulation of carotid baroreceptors in unanesthetized rats treated with desoxycorticosterone acetate caused highly significant decreases in solute content and osmolar concentration in the inner renal medulla. There was also a corresponding decrease in urine osmolality and a large increase in the excretion of sodium. In rats subjected to water diuresis, the changes in medullary tissue composition were similar but sodium excretion was very low, indicating that the natriuretic response was not a result of medullary "washout" per se. Renal denervation had no significant effect on medullary tissue composition and did not prevent the dissipation of the cortico-medullary concentration gradient following carotid baroreceptor stimulation. It is concluded that the changes in inner medullary composition are mediated by a humoral agent.     

The cellular localization of increased atrial natriuretic peptide mRNA and immunoreactivity in diabetic rat kidneys.

Increased intrarenal atrial natriuretic peptide (ANP) mRNA expression has been reported in several disorders. To further investigate the action of renal ANP, we need to elucidate the exact site of its alteration in diseased kidneys. ANP mRNA and ANP were detected by in situ hybridization and immunohistochemistry in the kidneys from five normal and five diabetic rats. Renal ANP mRNA in eight normal and nine diabetic rats was measured by RT-PCR with Southern blot hybridization. In normal and diabetic rats, the distribution of ANP mRNA and ANP-like peptide was mainly located in proximal, distal, and collecting tubules. However, diabetic rats had significant enhancement of ANP mRNA and ANP-immunoreactive staining in the proximal straight tubules, medullary thick ascending limbs, and medullary collecting ducts. ANP mRNA in the outer and inner medulla of nine diabetic rats increased 5.5-fold and 3.5-fold, but only 1.8-fold in the renal cortex. This preliminary study showed that ANP mRNA and ANP immunoreactivity in proximal straight tubules, medullary thick ascending limb, and medullary collecting ducts apparently increased in diabetic kidneys. These findings imply that ANP synthesis in these nephrons may involve in adaptations of renal function in diabetes.     

Effect of oxygen and solute on PGE and PGF production by rat kidney slices

Increasing oxygen from 5% to 95% resulted in an increased production of both PGE's and PGF's. The release of prostaglandins from slices of rat kidney cortex and outer and inner medulla was measured. Prostaglandin production was observed predominantly in the inner medulla, was close to the lower limit of detection in the outer medulla, and was undetectable in the cortex. Increasing oxygen concentration resulted in a threefold increase in inner medullary prostaglandin production. Synthesis at 95% O₂ was less at 2100 mOsm than at 300 mOsm, while synthesis at 5% O₂ was not affected by high solute concentration. The implications of these results with respect to kidney function are discussed.     

Membrane microdomain components of Histoplasma capsulatum yeast forms, and their role in alveolar macrophage infectivity

Analysis of membrane lipids of Histoplasma capsulatum showed that ~40% of fungal ergosterol is present in membrane microdomain fractions resistant to treatment with non-ionic detergent at 4°C. Specific proteins were also enriched in these fractions, particularly Pma1p a yeast microdomain protein marker (a plasma membrane proton ATPase), a 30kDa laminin-binding protein, and a 50kDa protein recognized by anti-α5-integrin antibody. To better understand the role of ergosterol-dependent microdomains in fungal biology and pathogenicity, H. capsulatum yeast forms were treated with a sterol chelator, methyl-beta-cyclodextrin (mβCD). Removal of ergosterol by mβCD incubation led to disorganization of ergosterol-enriched microdomains containing Pma1p and the 30kDa protein, resulting in displacement of these proteins from detergent-insoluble to -soluble fractions in sucrose density gradient ultracentrifugation. mβCD treatment did not displace/remove the 50kDa α5-integrin-like protein nor had effect on the organization of glycosphingolipids present in the detergent-resistant fractions. Ergosterol-enriched membrane microdomains were also shown to be important for infectivity of alveolar macrophages; after treatment of yeasts with mβCD, macrophage infectivity was reduced by 45%. These findings suggest the existence of two populations of detergent-resistant membrane microdomains in H. capsulatum yeast forms: (i) ergosterol-independent microdomains rich in integrin-like proteins and glycosphingolipids, possibly involved in signal transduction; (ii) ergosterol-enriched microdomains containing Pma1p and the 30kDa laminin-binding protein; ergosterol and/or the 30kDa protein may be involved in macrophage infectivity.     

DC-SIGN and influenza hemagglutinin dynamics in plasma membrane microdomains are markedly different

DC-SIGN, a Ca²⁺-dependent transmembrane lectin, is found assembled in microdomains on the plasma membranes of dendritic cells. These microdomains bind a large variety of pathogens and facilitate their uptake for subsequent antigen presentation. In this study, DC-SIGN dynamics in microdomains were explored with several fluorescence microscopy methods and compared with dynamics for influenza hemagglutinin (HA), which is also found in plasma membrane microdomains. Fluorescence imaging indicated that DC-SIGN microdomains may contain other C-type lectins and that the DC-SIGN cytoplasmic region is not required for microdomain formation. Fluorescence recovery after photobleaching measurements showed that neither full-length nor cytoplasmically truncated DC-SIGN in microdomains appreciably exchanged with like molecules in other microdomains and the membrane surround, whereas HA in microdomains exchanged almost completely. Line-scan fluorescence correlation spectroscopy indicated an essentially undetectable lateral mobility for DC-SIGN but an appreciable mobility for HA within their respective domains. Single-particle tracking with defined-valency quantum dots confirmed that HA has significant mobility within microdomains, whereas DC-SIGN does not. By contrast, fluorescence recovery after photobleaching indicated that inner leaflet lipids are able to move through DC-SIGN microdomains. The surprising stability of DC-SIGN microdomains may reflect structural features that enhance pathogen uptake either by providing high-avidity platforms and/or by protecting against rapid microdomain endocytosis.