Recent progress in histochemistry

The progress in discerning the structure and function of cells and tissues in health and disease has been achieved to a large extent by the continued development of new reagents for histochemistry, the improvement of existing techniques and new imaging techniques. This review will highlight some advancements made in these field

Expression of mutant Ins2C96Y results in enhanced tubule formation causing enlargement of pre-Golgi intermediates of CHO cells

Misfolded proteins are recognized by the protein quality control and eventually degraded by the ubiquitin-proteasome system. Previously, we demonstrated accumulation of a misfolded non-glycosylated protein, namely proinsulin, in enlarged pre-Golgi intermediates and dilated rough endoplasmic reticulum (ER) domains in pancreatic β-cells of Akita mice. In order to exclude effects possibly due to coexisting wild type and mutant proinsulin in pancreatic β-cells, CHO cells expressing singly wild type or mutant C96Y proinsulin 2 were now analyzed by electron microscopic morphometry and immunogold labeling as well as serial section 3D analysis. We found a significant increase in volume density of pre-Golgi intermediates in CHO Ins2C96Y cells which was principally due to an increase of its tubular elements, and no significant changes of the ER. The average diameter of the pre-Golgi intermediates of CHO Ins2C96Y cells was about twice that of CHO Ins2wt cells. The enlarged pre-Golgi intermediates and the ER of CHO Ins2C96Y cells were positive for proinsulin, which was not detectable in the significantly enlarged Golgi cisternal stack. Treatment of CHO Ins2C96Y cells with proteasome inhibitors resulted in the formation of proinsulin-containing aggresomes. We conclude that misfolded proinsulin causes enlargement of pre-Golgi intermediates which indicates their involvement in protein quality contro

Specialized expression of simple O-glycans along the rat kidney nephron

Glycosyltransferases can exhibit tissue-specific expression. By histochemistry glycosyltransferases and their products can be localized to specific cell types in organs of complex cellular composition. We have applied the lectin Amaranthin, having a nominal specificity for Galβ1,3GalNAcR and Neu5Ac2,3Galβ1,3GalNAcα-R, and a monoclonal antibody raised against Galβ1,3GalNAcαR to examine the distribution of these simple O-glycans in adult rat kidney. The monoclonal antibody stained ascending thin limbs of Henle, distal convoluted tubules, and collecting ducts of cortex and outer medulla. Remarkably, the ascending thick limb of Henle, located between ascending thin limb and distal convoluted tubules, was unreactive. However, Amaranthin staining was detectable in ascending thick limbs of Henle, in addition to the structures positive with the monoclonal antibody. In kidney extracts, two bands of approximately 160 kDa and >210 kDa were reactive with both Amaranthin and the monoclonal antibody. One band at ∼200 kDa, and a smear at ∼100 kDa, were reactive only with Amaranthin. Our data show that in rat kidney simple O-linked glycans are expressed in a highly specialized manner along the renal tubule and can be detected only on a few glycoproteins. This may reflect a cell-type-specific expression of the corresponding glycosyltransferase

Large protein complexes retained in the ER are dislocated by non-COPII vesicles and degraded by selective autophagy

Multisubunit protein complexes are assembled in the endoplasmic reticulum (ER). Existing pools of single subunits and assembly intermediates ensure the efficient and rapid formation of complete complexes. While being kinetically beneficial, surplus components must be eliminated to prevent potentially harmful accumulation in the ER. Surplus single chains are cleared by the ubiquitin-proteasome system. However, the fate of not secreted assembly intermediates of multisubunit proteins remains elusive. Here we show by high-resolution double-label confocal immunofluorescence and immunogold electron microscopy that naturally occurring surplus fibrinogen Aα-γ assembly intermediates in HepG2 cells are dislocated together with EDEM1 from the ER to the cytoplasm in ER-derived vesicles not corresponding to COPII-coated vesicles originating from the transitional ER. This route corresponds to the novel ER exit path we have previously identified for EDEM1 (Zuber etal. Proc Natl Acad Sci USA 104:4407-4412, 2007). In the cytoplasm, detergent-insoluble aggregates of fibrinogen Aα-γ dimers develop that are targeted by the selective autophagy cargo receptors p62/SQSTM1 and NBR1. These aggregates are degraded by selective autophagy as directly demonstrated by high-resolution microscopy as well as biochemical analysis and inhibition of autophagy by siRNA and kinase inhibitors. Our findings demonstrate that different pathways exist in parallel for ER-to-cytoplasm dislocation and subsequent proteolytic degradation of large luminal protein complexes and of surplus luminal single-chain proteins. This implies that ER-associated protein degradation (ERAD) has a broader function in ER proteostasis and is not limited to the elimination of misfolded glycoprotein