Christoph Greb

Apical sorting by galectin-3-dependent glycoprotein clustering

Epithelial cells are characterized by their polarized organization based on an apical membrane that is separated from the basolateral membrane domain by tight junctions. Maintenance of this morphology is guaranteed by highly specific sorting machinery that separates lipids and proteins into different carrier populations for the apical or basolateral cell surface. Lipid‐raft‐independent apical carrier vesicles harbour the beta‐galactoside‐binding lectin galectin‐3, which interacts directly with apical cargo in a glycan‐dependent manner. These glycoproteins are mistargeted to the basolateral membrane in galectin‐3‐depleted cells, dedicating a central role to this lectin in raft‐independent sorting as apical receptor. Here, we demonstrate that high‐molecular‐weight clusters are exclusively formed in the presence of galectin‐3. Their stability is sensitive to increased carbohydrate concentrations, and cluster formation as well as apical sorting are perturbed in glycosylation‐deficient Madin‐Darby canine kidney (MDCK) II cells. Together, our data suggest that glycoprotein cross‐linking by galectin‐3 is required for apical sorting of non‐raft‐associated cargo.

Trafficking of galectin-3 through endosomal organelles of polarized and non-polarized cells.

In epithelial cells, the β-galactoside-binding lectin galectin-3 mediates the non-raft-dependent glycoprotein targeting to the apical membrane domain. In this study, we aimed to identify intracellular compartments involved in the trafficking of galectin-3. By studying fluorescent fusion proteins in living cells, we could show that galectin-3 accumulates intracellularly in acidified endosomes. Total internal reflection fluorescence microscopy studies of the apical surface of polarized MDCK cells revealed that galectin-3 is enriched in tubular and vesicular Rab11-positive recycling endosomes in the vicinity of the apical cell surface. These endosomal organelles are candidate compartments for the association between galectin-3 and exocytic apical cargo.

pH-Dependent Recycling of Galectin-3 at the Apical Membrane of Epithelial Cells

The β‐galactoside binding protein galectin‐3 is highly expressed in a variety of epithelial cell lines. Polarized MDCK cells secrete this lectin predominantly into the apical medium by non‐classical secretion. Once within the apical extracellular milieu, galectin‐3 can re‐enter the cell followed by passage through endosomal organelles and modulate apical protein sorting. Here, we could show that galectin‐3 is internalized by non‐clathrin mediated endocytosis. Within endosomal organelles this pool associates with newly synthesized neurotrophin receptor in the biosynthetic pathway and assists in its membrane targeting. This recycling process is accompanied by transient interaction of galectin‐3 with detergent insoluble membrane microdomains in a lactose‐ and pH‐dependent manner. Moreover, in the presence of lactose, apical sorting of the neurotrophin receptor is affected following endosomal deacidification. Taken together, our results suggest that internalized galectin‐3 directs the subcellular targeting of apical glycoproteins by membrane recycling.

Changes in the expression and subcellular distribution of galectin-3 in clear cell renal cell carcinoma

BackgroundClear cell renal cell carcinoma, a solid growing tumor, is the most common tumor in human kidney. Evaluating the usefulness of β-galactoside binding galectin-3 as a diagnostic marker for this type of cancer could open avenues for preventive and therapeutic strategies by employing specific inhibitors of the lectin. To study a putative correlation between the extent of galectin-3 and the development of clear cell renal cell carcinoma, we monitored the quantity and distribution of this lectin in tissue samples from 39 patients.MethodsGalectin-3 concentrations in normal, intermediate and tumor tissues were examined by immunofluorescence microscopy and on immunoblots with antibodies directed against galectin-3 and renal control proteins. The cell nuclei were isolated to determine quantities of galectin-3 that were transferred into this compartment in normal or tumor samples.ResultsImmunofluorescence data revealed a mosaic pattern of galectin-3 expression in collecting ducts and distal tubules of normal kidney. Galectin-3 expression was significantly increased in 79% of tumor samples as compared to normal tissues. Furthermore, we observed an increase in nuclear translocation of the lectin in tumor tissues.ConclusionsOur data indicate that changes in the cellular level of galectin-3 correlate with the development of clear cell renal cell carcinoma, which is in line with previously published data on this specific type of tumor. In most of these studies the lectin tends to be highly expressed in tumor tissues. Furthermore, this study suggests that the increase in the proportion of galectin-3 affects the balance from a cytosolic distribution towards translocation into the nucleus.

The anaphylatoxin C5a, a new parameter in the diagnosis of renal allograft rejection

Abstract  In the underlying study the diagnostic value of the anaphylatoxin CSa was evaluated in kidney transplantation. In 49 transplant patients the following parameters were measured daily for a mean period of 25.1 days: plasma a [P‐CSa], urine a [U‐CSa], serum amyloid A (SAA], serum neopterin [S‐NEOP] and urine neopterin [U‐NEOP]. Sensitivity, specificity and day of first significant parameter increase (exceeding a cut‐off level of > 50 YO) were evaluated retrospectively during 30 periods of rejection and 30 periods of stable graft function. U‐ a was the parameter with the highest sensitivity (84 %) and specificity (84 %), increasing in the mean 1.3 days before clinical diagnosis of rejection. Sensitivity and specificity of the other markers was lower: SAA 77 % and 77 %, U‐NEOP 68 % and 65 %, S‐NEOP 45 % and 77 %, and P‐ a 45 % and 48 %, respectively. During four instances of cy‐tomegalovirus disease extremely high U‐NEOP (≥= 1520 ± 518 μmol/mol creatinine) and slightly increased P‐ a levels (≥= 1.5 ± 1.4 ng/ml) occurred. Elevated urinary excretion of a seems to be a reliable and early marker of renal allograft rejection. In combination with SAA and U‐NEOP, the daily assessment of U‐ a differentiates between viral infection and allograft rejection.

The Large GTPase Mx1 Is Involved in Apical Transport in MDCK Cells

In epithelial cells apical proteins are transported by specific transport carriers to the correct membrane domain. The composition of these carriers is heterogeneous and comprises components such as motor proteins, annexins, lectins, Rab GTPases and cargo molecules. Here, we provide biochemical and fluorescence microscopic data to show that the dynamin‐related large GTPase Mx1 is a component of post‐Golgi vesicles carrying the neurotrophin receptor p75NTR. Moreover, siRNA‐mediated depletion of Mx1 significantly decreased the transport efficiency of apical proteins in MDCK cells. In conclusion, Mx1 plays a crucial role in the delivery of cargo molecules to the apical membrane of epithelial cells.

TIRF-Mikroskopie auf den Kopf gestellt

ZusammenfassungUnter Anwendung der TIRF-Mikroskopie lassen sich auch Strukturen an apikalen Oberflächen polarer Epithelzellen darstellen. Somit kann ein neuer Einblick in den Aufbau dieses charakteristischen Zellpols gewonnen werden, der schließich zur Aufklärung apikaler Proteintransportprozesse führen soll.AbstractApplication of TIRF-microscopy allows the visualization of structures at the apical surface of polarized epithelial cells. Hence, the approach reveals new insights into the composition of this characteristic cell pole that elucidate processes in apical protein trafficking.