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Homozygous Apolipoprotein L1 (APOL1) variants G1 and G2 cause APOL1-mediated kidney disease, purportedly acting as surface cation channels in podocytes. APOL1-G0 exhibits various single nucleotide polymorphisms, most commonly haplotype E150K, M228I and R255K ("KIK"; the Reference Sequence is "EMR"), whereas variants G1 and G2 are mostly found in a single "African" haplotype background ("EIK"). Several labs reported cytotoxicity with risk variants G1 and G2 in KIK or EIK background haplotypes, but used HEK-293 cells and did not verify equal surface expression. To see if haplotype matters in a more relevant cell type, we induced APOL1-G0, G1 and G2 EIK, KIK and EMR at comparable surface levels in immortalized podocytes. G1 and G2 risk variants (but not G0) caused dose-dependent podocyte death within 48h only in their native African EIK haplotype and correlated with K + conductance (thallium FLIPR). We ruled out differences in localization and trafficking, except for possibly greater surface clustering of cytotoxic haplotypes. APOL1 surface expression was required, since Brefeldin A rescued cytotoxicity; and cytoplasmic isoforms vB3 and vC were not cytotoxic. Thus, APOL1-EIK risk variants kill podocytes in a dose and haplotype-dependent manner (as in HEK-293 cells), whereas unlike in HEK-293 cells the KIK risk variants did not., (© 2024. The Author(s).)

Background: APOL1 is found in human kidney podocytes and endothelia. Variants G1 and G2 of the APOL1 gene account for the high frequency of nondiabetic CKD among African Americans. Proposed mechanisms of kidney podocyte cytotoxicity resulting from APOL1 variant overexpression implicate different subcellular compartments. It is unclear where endogenous podocyte APOL1 resides, because previous immunolocalization studies utilized overexpressed protein or commercially available antibodies that crossreact with APOL2. This study describes and distinguishes the locations of both APOLs., Methods: Immunohistochemistry, confocal and immunoelectron microscopy, and podocyte fractionation localized endogenous and transfected APOL1 using a large panel of novel APOL1-specific mouse and rabbit monoclonal antibodies., Results: Both endogenous podocyte and transfected APOL1 isoforms vA and vB1 (and a little of isoform vC) localize to the luminal face of the endoplasmic reticulum (ER) and to the cell surface, but not to mitochondria, endosomes, or lipid droplets. In contrast, APOL2, isoform vB3, and most vC of APOL1 localize to the cytoplasmic face of the ER and are consequently absent from the cell surface. APOL1 knockout podocytes do not stain for APOL1, attesting to the APOL1-specificity of the antibodies. Stable re-transfection of knockout podocytes with inducible APOL1-G0 , -G1 , and - G2 showed no differences in localization among variants., Conclusions: APOL1 is found in the ER and plasma membrane, consistent with either the ER stress or surface cation channel models of APOL1-mediated cytotoxicity. The surface localization of APOL1 variants potentially opens new therapeutic targeting avenues., (Copyright © 2020 by the American Society of Nephrology.)

Melanosomes are lysosome-related organelles that coexist with lysosomes in mammalian pigment cells. Melanosomal and lysosomal membrane proteins share similar sorting signals in their cytoplasmic tail, raising the question how they are segregated. We show that in control melanocytes, the melanosomal enzymes tyrosinase-related protein 1 (Tyrp1) and tyrosinase follow an intracellular Golgi to melanosome pathway, whereas in the absence of glycosphingolipids, they are observed to pass over the cell surface. Unexpectedly, the lysosome-associated membrane protein 1 (LAMP-1) and 2 behaved exactly opposite: they were found to travel through the cell surface in control melanocytes but followed an intracellular pathway in the absence of glycosphingolipids. Chimeric proteins having the cytoplasmic tail of Tyrp1 or tyrosinase were transported like lysosomal proteins, whereas a LAMP-1 construct containing the lumenal domain of Tyrp1 localized to melanosomes. In conclusion, the lumenal domain contains sorting information that guides Tyrp1 and probably tyrosinase to melanosomes by an intracellular route that excludes lysosomal proteins and requires glucosylceramide.

Glycosphingolipids are controlled by the spatial organization of their metabolism and by transport specificity. Using immunoelectron microscopy, we localize to the Golgi stack the glycosyltransferases that produce glucosylceramide (GlcCer), lactosylceramide (LacCer), and GM3. GlcCer is synthesized on the cytosolic side and must translocate across to the Golgi lumen for LacCer synthesis. However, only very little natural GlcCer translocates across the Golgi in vitro. As GlcCer reaches the cell surface when Golgi vesicular trafficking is inhibited, it must translocate across a post-Golgi membrane. Concanamycin, a vacuolar proton pump inhibitor, blocks translocation independently of multidrug transporters that are known to translocate short-chain GlcCer. Concanamycin did not reduce LacCer and GM3 synthesis. Thus, GlcCer destined for glycolipid synthesis follows a different pathway and transports back into the endoplasmic reticulum (ER) via the late Golgi protein FAPP2. FAPP2 knockdown strongly reduces GM3 synthesis. Overall, we show that newly synthesized GlcCer enters two pathways: one toward the noncytosolic surface of a post-Golgi membrane and one via the ER toward the Golgi lumen LacCer synthase.

Endocytosis is crucial for various aspects of cell homeostasis. Here, we show that proapoptotic death receptors (DRs) trigger selective destruction of the clathrin-dependent endocytosis machinery. DR stimulation induced rapid, caspase-mediated cleavage of key clathrin-pathway components, halting cellular uptake of the classic cargo protein transferrin. DR-proximal initiator caspases cleaved the clathrin adaptor subunit AP2alpha between functionally distinct domains, whereas effector caspases processed clathrin's heavy chain. DR5 underwent ligand-induced, clathrin-mediated endocytosis, suggesting that internalization of DR signaling complexes facilitates clathrin-pathway targeting by caspases. An endocytosis-blocking, temperature-sensitive dynamin-1 mutant attenuated DR internalization, enhanced caspase stimulation downstream of DRs, and increased apoptosis. Thus, DR-triggered caspase activity disrupts clathrin-dependent endocytosis, leading to amplification of programmed cell death.

ErbB2 is a transmembrane tyrosine kinase whose surface overexpression is linked to tumorigenesis and poor prognosis in breast cancer patients. Two models have emerged that account for the high surface distribution of ErbB2. In one model, the surface pool is dynamic and governed by a balance between endocytosis and recycling, whereas in the other it is retained, static, and excluded from endocytosis. These models have contrasting implications for how ErbB2 exerts its biological function and how cancer therapies might down-regulate surface ErbB2, such as the antibody trastuzumab (Herceptin) or the Hsp90 inhibitor geldanamycin. Little is known, however, about how these treatments affect ErbB2 endocytic trafficking. To investigate this issue, we examined breast carcinoma cells by immunofluorescence and quantitative immunoelectron microscopy and developed imaging and trafficking kinetics assays using cell surface fluorescence quenching. Surprisingly, trastuzumab does not influence ErbB2 distribution but instead recycles passively with internalized ErbB2. By contrast, geldanamycin down-regulates surface ErbB2 through improved degradative sorting in endosomes exclusively rather than through increased endocytosis. These results reveal substantial dynamism in the surface ErbB2 pool and clearly demonstrate the significance of endosomal sorting in the maintenance of ErbB2 surface distribution, a critical feature of its biological function.

To delineate the role of the melanocyte lineage-specific protein Melan-A/MART-1 in melanogenic functions, a set of biochemical and microscopical studies was performed. Biochemical analysis revealed that Melan-A/MART-1 is post-translationally acylated and undergoes a rapid turnover in a pigmented melanoma cell line. Immunofluorescence and immunoelectron microscopy analyses indicated that Melan-A/MART-1 is mainly located in the Golgi area and only partially colocalizes with melanosomal proteins. Quantitative immunoelectron microscopy showed that the highest proportion of the cellular content of Melan-A/MART-1 was found in small vesicles and tubules throughout the cell, whereas the concentration was maximal in the Golgi region, particularly the trans-Golgi network. Substantial labeling was also present on melanosomes, endosomes, ER, nuclear envelope, and plasma membrane. In early endosomes, Melan-A was enriched in areas of the limiting membrane covered by a bi-layered coat, a structural characteristic of melanosomal precursor compartments. Upon melanosome maturation, Melan-A concentration decreased and its predominant localization shifted from the limiting membrane to internal vesicle membranes. In conjunction with its acylation, the high expression levels of Melan-A in the trans-Golgi network, in dispersed vesicles, and on the limiting membrane of premelanosomes indicate that the protein may play a role during the early stage of melanosome biogenesis.

To study the cell cleavage pattern in experimentally treated Xenopus laevis blastulae, we devised a method to visualize all cell nuclei, whether in interphase or in a mitotic phase, in whole-mount embryos using confocal laser scanning microscopy. Optimal staining conditions were defined for the recently commercialized cyanine nucleic acid stain TO-PRO-3, which is excited by a 647-nm laser beam and fluoresces in the far red of the spectrum. This is beyond the spectral range of autofluorescence caused by most biomolecules and, in particular, by the high amount of yolk granules in these embryos. The quality of the TO-PRO-3 image was compared to that after nuclear staining with BOBO-3, another cyanine dye that fluoresces at slightly shorter wavelengths. In the proposed procedure, special attention is paid to permeabilization of the membranes to the dyes and to bleaching of the natural pigment of the embryos with maximal preservation of cellular and nuclear structures. Because of its emission maximum at 661 nm, TO-PRO-3 is a promising nuclear stain for specimens with special background problems and for multicolor fluorescence microscopy.

In an attempt to understand better the directional differences in conduction velocity in the rabbit sinoatrial node, a possible conductive role of the abundant connective tissue surrounding the myocytes has been investigated. In particular, starting from the finding of communicating junctions between heart muscle cells and fibroblasts in tissue culture, heterologous gap junctions were searched for in thin sections of the rabbit sinoatrial node. Within and at the edge of nodal cell clusters, fibroblasts often show thin sheet-like extensions parallel to the surface of myocytes. In contrast to the intimately contacting myocytes, fibroblast extensions are kept separated from the myocytes by the basement membrane of the latter. Besides some rare undefined membrane appositions a single tiny gap junction-like structure was found between a fibroblast and a myocyte in a tissue area in which the calculated number of gap junctions between myocytes amounts from 1.10(4) to 3.10(4). Yet, fibroblasts are linked together regularly by small gap junctions containing a wider gap than the junctions between the myocytes (1.4 +/- 0.4 nm vs. 1.0 +/- 0.4 nm, resp., P less than 0.05). As an alternative to direct electrical coupling, the possibility of interaction between fibroblasts and nodal cells by capacitive coupling has been considered. Model calculations based on the reconstruction of some fibroblast extensions parallel to nodal cells show that the current which can be transmitted from discharging nodal cells to fibroblasts is negligible. It is concluded that fibroblasts do not participate in the impulse conduction within the sinoatrial node. The origin of the directional differences in conduction velocity in the sinoatrial node must be found in the spatial arrangement of the myocytes and the distribution of the gap junctions between these cells only.

Isolated rat hearts were subjected to increasing periods of hypoxia with or without subsequent reoxygenation and the gap-junctional particle configuration was followed quantitatively. Irregular contractions were prevented by K(+)-arrest; glucose, counteracting the effects of hypoxia, was omitted. Hyperkalemia alone and a maximum of 20 min of hypoxia do not produce reorganization of the gap-junctional particles normally forming multiple hexagonally packed arrays separated by smooth aisles. After 30 min of hypoxia, the aisles disappear in a proportion of the junctions, thereby increasing the particle density from 9400 +/- 800/microns2 to 10,200 +/- 900/microns2. After 40 min of hypoxia, the normal configuration is no longer found and numerous junctions are arranged as uninterrupted hexagonal lattices. The particles are further condensed to 11,600 +/- 900/microns2. Following reoxygenation after both 30 and 40 min of hypoxia, the proportion of crystalline gap junctions dramatically augments and the mean particle density has further increased significantly. Corresponding thin sections show irreversible cell damage. When reoxygenation is performed with a control solution containing normal levels of K+ and glucose, the particle density does not increase substantially in comparison to the respective 30- and 40-min hypoxic periods. In both groups, the gap junctions display either a normal, a crystalline or an intermediate configuration with crystalline margins and loose centers. The gap-junctional reorganization during hypoxia essentially represents a particle condensation, while the mean center-to-center distances between the particles and pits remain constant. Furthermore, the reappearance of normal gap junctions after reoxygenation appears to depend on glucose availability.

The effect of membrane depolarization was investigated on gap junctions from isolated rat hearts perfused with a modified Krebs-Henseleit solution containing 16 mM K+. After freeze-fracturing, the configuration of the junctional particles in the ventricular myocardium was analysed by measurements of connexon densities and centre-to-centre distances between neighbouring particles. Both in control and hyperkalemic tissue, the gap junctions occur on the intercalated discs as round or oval aggregates of connexons which are closely and regularly packed in small, criss-cross-oriented arrays separated by particle-free aisles. Within the arrays, the mean (+/- SD) centre-to-centre distances between particles from control and hyperkalemic tissue, i.e. 9.17 +/- 1.52 and 9.15 +/- 1.51 mm, respectively, are not significantly different. Similarly, comparison of particle densities after control and high-K+ perfusion, i.e. 8,490 +/- 600 and 8,420 +/- 620 particles/microns 2, respectively, reveals no difference in the proportion of the particle arrays to the empty aisles. The apparently unaltered gap junctional morphology after depolarization of the sarcolemma by high-K+ perfusion provides support for the electrophysiological finding that the conductance of cardiac gap junctions is insensitive to membrane potential.

In view of the presumed involvement of gap junctions in the coordination of metabolic activities, the influence of cAMP as a regulatory signal of cell metabolism on gap junctions of hepatocytes has been examined. Male rats received two intraperitoneal doses of 10 mg dibutyryl cAMP/100 g body weight with a time interval of 2.5 h and were decapitated 2.5 h later. After this 5-h interval, analysis of freeze-fracture replicas of fixed liver tissue revealed an increase in the mean (+/- SEM) gap-junctional membrane portion on the lateral hepatocyte membranes from 0.049 +/- 0.003 (n = 66) in controls to 0.061 +/- 0.003 (n = 70) in treated rats, while the configuration of the connexons appeared unaltered. This effect could not be reinforced by prior administration of aminophylline: the relative gap-junctional area is similarly extended from 0.054 +/- 0.003 (n = 126) in the control group to 0.065 +/- 0.004 (n = 105) in the experimental animals. Probing for the time course of the junctional response, a group of rats was sacrificed 3 h after the onset of treatment. Already within this time, the gap-junctional area is augmented from 0.042 +/- 0.004 (n = 63) in the concurrent controls to 0.069 +/- 0.006 (n = 42) in the treated rats. These statistically significant increases in area may suggest a stimulating effect of cAMP on gap junctions of hepatocytes in vivo.

A method for cleaning freeze-fracture replicas is elaborated which avoids their rolling up and fragmentating during successive steps. Immediately after freeze-fracturing, the replicated tissue is slowly thawed from 77 K to room temperature on solid methanol. Safe passage through the subsequent cleaning steps is facilitated by using solutions of gradually increasing surface tension. After at least 24 h of fixation in methanol, the tissue is digested in half-strength bleach containing 5% ethanol. Following further cleaning in full-strength bleach, the remaining organic material attached to the replica is dissolved overnight at room temperature in 50% saturated sodium hydroxide.

Particles and pits of freeze-fractured gap junctions are considered as complementary structures despite the frequent observations of more regular and closer spacings of pits, ascribed to plastic deformation of particle arrays. Recently, however, the noncomplementarity of pits and particles in Purkinje fibers has been reported. To ascertain the relationship between both structures, gap junctions from fixed, cryoprotected liver and myocardium were investigated using spacing and density measurements and complementary replicas. In hepatocyte gap junctions, the center-to-center distances (mean +/- SD) among pits, 9.57 +/- 1.49 nm, and particles, 9.70 +/- 1.77 nm, are not significantly different. Density determinations yielded a slightly higher value for the pits, (11,510 +/- 830)/microns 2, than for the particles, (11,230 +/- 950)/microns 2. In the myocardium, the spacing of the regularly arrayed pits, 9.55 +/- 1.33 nm, barely exceeds the value of 9.44 +/- 1.62 nm for the particles, which show some clustering. However, the packing density for the pits, (10,090 +/- 740)/microns 2, appears a little higher than that of the particles, (9,890 +/- 920)/microns 2. As density and spacing measurements provided no decisive answers, the positions of individual pits and particles of complementary junctional faces were recorded on transparent sheets and compared. In this fashion, a one-to-one correspondence between particles and pits could be established, while small discrepancies may be attributed to plastic deformation. Moreover, the co-linearity of pits and particles may be suggested by the observation of a platinum grain in the center of many pits.