Sergei I. Bannykh

COPII-dependent export of cystic fibrosis transmembrane conductance regulator from the ER uses a di-acidic exit code

Cystic fibrosis (CF) is a childhood hereditary disease in which the most common mutant form of the CF transmembrane conductance regulator (CFTR) ΔF508 fails to exit the endoplasmic reticulum (ER). Export of wild-type CFTR from the ER requires the coat complex II (COPII) machinery, as it is sensitive to Sar1 mutants that disrupt normal coat assembly and disassembly. In contrast, COPII is not used to deliver CFTR to ER-associated degradation. We find that exit of wild-type CFTR from the ER is blocked by mutation of a consensus di-acidic ER exit motif present in the first nucleotide binding domain. Mutation of the code disrupts interaction with the COPII coat selection complex Sec23/Sec24. We propose that the di-acidic exit code plays a key role in linking CFTR to the COPII coat machinery and is the primary defect responsible for CF in ΔF508-expressing patients.

Regulation of Sar1 NH2 terminus by GTP binding and hydrolysis promotes membrane deformation to control COPII vesicle fission

The mechanisms by which the coat complex II (COPII) coat mediates membrane deformation and vesicle fission are unknown. Sar1 is a structural component of the membrane-binding inner layer of COPII (Bi, X., R.A. Corpina, and J. Goldberg. 2002. Nature. 419:271–277). Using model liposomes we found that Sar1 uses GTP-regulated exposure of its NH2-terminal tail, an amphipathic peptide domain, to bind, deform, constrict, and destabilize membranes. Although Sar1 activation leads to constriction of endoplasmic reticulum (ER) membranes, progression to effective vesicle fission requires a functional Sar1 NH2 terminus and guanosine triphosphate (GTP) hydrolysis. Inhibition of Sar1 GTP hydrolysis, which stabilizes Sar1 membrane binding, resulted in the formation of coated COPII vesicles that fail to detach from the ER. Thus Sar1-mediated GTP binding and hydrolysis regulates the NH2-terminal tail to perturb membrane packing, promote membrane deformation, and control vesicle fission.

Non-conventional Trafficking of the Cystic Fibrosis Transmembrane Conductance Regulator through the Early Secretory Pathway

The mechanism(s) of cystic fibrosis transmembrane conductance regulator (CFTR) trafficking from the endoplasmic reticulum (ER) through the Golgi apparatus, the step impaired in individuals afflicted with the prevalent CFTR-ΔF508 mutation leading to cystic fibrosis, is largely unknown. Recent morphological observations suggested that CFTR is largely absent from the Golgi in situ (Bannykh, S. I., Bannykh, G. I., Fish, K. N., Moyer, B. D., Riordan, J. R., and Balch, W. E. (2000)Traffic 1, 852–870), raising the possibility of a novel trafficking pathway through the early secretory pathway. We now report that export of CFTR from the ER is regulated by the conventional coat protein complex II (COPII) in all cell types tested. Remarkably, in a cell type-specific manner, processing of CFTR from the core-glycosylated (band B) ER form to the complex-glycosylated (band C) isoform followed a non-conventional pathway that was insensitive to dominant negative Arf1, Rab1a/Rab2 GTPases, or the SNApREceptor (SNARE) component syntaxin 5, all of which block the conventional trafficking pathway from the ER to the Golgi. Moreover, CFTR transport through the non-conventional pathway was potently blocked by overexpression of the late endosomal target-SNARE syntaxin 13, suggesting that recycling through a late Golgi/endosomal system was a prerequisite for CFTR maturation. We conclude that CFTR transport in the early secretory pathway can involve a novel pathway between the ER and late Golgi/endosomal compartments that may influence developmental expression of CFTR on the cell surface in polarized epithelial cells.

A Di-acidic (DXE) Code Directs Concentration of Cargo during Export from the Endoplasmic Reticulum

Efficient export of vesicular stomatitis virus glycoprotein (VSV-G), a type I transmembrane protein, from the endoplasmic reticulum requires a di-acidic code (DXE) located in the cytosolic carboxyl-terminal tail (Nishimura, N., and Balch, W. E. (1997) Science 277, 556–558). Mutation of the DXE code by mutation to AXA did not prevent VSV-G recruitment to pre-budding complexes formed in the presence of the activated form of the Sar1 and the Sec23/24 complex, components of the COPII budding machinery. However, the signal was required at a subsequent concentration step preceding vesicle fission. By using green fluorescence protein-tagged VSV-G to image movement in a single cell, we found that VSV-G lacking the DXE code fails to be concentrated into COPII vesicles. As a result, the normal 5–10-fold increase in the steady-state concentration of VSV-G in downstream pre-Golgi intermediates and Golgi compartments was lost. These results demonstrate for the first time that inactivation of the DXE signal uncouples early cargo selection steps from concentration into COPII vesicles. We propose that two sequential steps are required for efficient export from the endoplasmic reticulum.

Getting into the Golgi

The intermediate compartment residing between the endoplasmic reticulum (ER) and the Golgi is now recognized to be a dynamic structure that captures cargo released from the ER in COPII vesicular carriers and promotes recycling by COPI vesicular carriers. These and other findings now provide compelling evidence for the importance of this intermediate in balancing anterograde and retrograde flow through the early secretory pathway and in the formation and maintenance of the Golgi stack.

Differential Regulation of Exocytosis by Calcium and CAPS in Semi-Intact Synaptosomes

Using a novel approach to measure exocytosis in vitro from semi-intact synaptosomes, we establish that the Ca2+-dependent release of glutamate requires cytosolic factors for mobilization from the reserve pool. The cytosolic activity for glutamate release was not satisfied by CAPS, a soluble component required for norepinephrine (NE) release. Moreover, the CAPS-independent glutamate release from synaptic vesicles (SVs) was 200-fold less sensitive to Ca2+ than that required for dense core vesicles (DCVs). The differential regulation of exocytosis by CAPS, Ca2+, and potential novel cytosolic factor(s) suggests that the docking and fusion machinery controlling DCVs has diverged from that regulating glutamate-containing SVs.

Traffic Pattern of Cystic Fibrosis Transmembrane Regulator through the Early Exocytic Pathway

The pathway of transport of the cystic fibrosis transmembrane regulator (CFTR) through the early exocytic pathway has not been examined. In contrast to most membrane proteins that are concentrated during export from the ER and therefore readily detectable at elevated levels in pre‐Golgi intermediates and Golgi compartments, wild‐type CFTR could not be detected in these compartments using deconvolution immunofluorescence microscopy. To determine the basis for this unusual feature, we analyzed CFTR localization using quantitative immunoelectron microscopy (IEM). We found that wild‐type CFTR is present in pre‐Golgi compartments and peripheral tubular elements associated with the cis and trans faces of the Golgi stack, albeit at a concentration 2‐fold lower than that found in the endoplasmic reticulum (ER). ΔF508 CFTR, a mutant form that is not efficiently delivered to the cell surface and the most common mutation in cystic fibrosis, could also be detected at a reduced concentration in pre‐Golgi intermediates and peripheral cis Golgi elements, but not in post‐Golgi compartments. Our results suggest that the low level of wild‐type CFTR in the Golgi region reflects a limiting step in selective recruitment by the ER export machinery, an event that is largely deficient in ΔF508. We raise the possibility that novel modes of selective anterograde and retrograde traffic between the ER and the Golgi may serve to regulate CFTR function in the early secretory compartments.

Oligodendroglial-specific transcriptional factor SOX10 is ubiquitously expressed in human gliomas.

SummaryThe two most common types of gliomas: astrocytoma and oligodendroglioma are distinguished based on their morphologic similarities to mature astrocytes and oligodendroglia. Whereas prototypical examples of the tumors have distinct pathogenetic and prognostic differences, the majority of the gliomas falls in the intermediate category and their distinction is problematic. The transcriptional factor SOX10 is one of the key determinants of oligodendroglial differentiation. We applied immunohistochemistry to analyze whether the expression of SOX10 can differentiate astrocytoma and oligodendroglioma. The majority of oligodendrogliomas, but also a large fraction of astrocytomas, including the least differentiated glioblastomas, expressed SOX10, albeit at lower levels. Comparison with 1p and 19q deletion status by FISH analysis also revealed no obvious associations. High levels of expression were also found in pilocytic astrocytoma, consistent with recent studies suggesting that pilocytic astrocytomas have greater overlap with oligodendroglial than astrocytic tumors. Our data raise a possibility that histogenesis of gliomas have more common features than previously anticipated.

Fetal Obstructive Uropathy: Patterns of Renal Pathology

Fetal obstructive uropathy (FOU) is characterized by obstruction of the urethra, renal anomalies, ureterovesical dilatation, oligohydramnios, cryptorchidism, and abdominal muscle wall changes. The main objective of the present study was to better understand the relationship between FOU and renal pathology using a series of 15 male autopsy cases. A total of 11 cases with patent anus and 4 with imperforate anus were analyzed. Of the first group, most cases showed obstruction at the level of prostatic urethra. Seven cases showed obstruction at the level of the prostatic urethra and histologic study revealed scarring and partial or complete absence of the prostate, while in the remaining four cases the prostate was present. Of the cases with imperforate anus, two showed obstruction at the level of prostatic urethra, one showed posterior urethral valves, and one was obstructed at the proximal urethra. In all cases the kidneys showed mixed (dysplastic and cystic) changes with no significant differences between the two groups. An inverse correlation was observed between degree of renal dysplasia and gestational age, whereas the opposite was true for cystic changes. Distal and collecting tubules were more intensely immunoreactive to the anti-cytokeratin antibody when compared to proximal tubules. Moreover, anti-cytokeratin immunoreactivity was more prominent in tubules displaying cystic dilatation. DNA fragmentation analysis of renal tissue revealed a higher apoptosis of mesenchymal and tubular cells in the FOU cases, compared to gestational aged–matched controls. These results suggest that renal anomalies in FOU might be related to the gestational age at which the injury occurred and to the duration of the obstruction.

Aberrant Pax1 and Pax9 expression in Jarcho‐Levin syndrome: Report of two Caucasian siblings and literature review

We report two consecutive Caucasian male siblings of nonconsanguineous parents autopsied at 22 and 13 weeks gestational age both with prenatal diagnosis of Jarcho‐Levin syndrome (JLS). Segmentation anomalies of the vertebrae and ribs encompass a spectrum of syndromes with or without associated anomalies of other developmental fields, and include spondylothoracic dysostosis (STD), JLS, Casamassima‐Morton‐Nance (CMN) syndrome, and spondylocostal dysostosis (SCD), among others. In both these new JLS cases the autopsies confirmed that there were severe developmental alterations in the thoracic and vertebral skeleton (including “crab‐like” thorax), accompanied in the older fetus by renal defects. Because vertebral development is controlled by a limited number of master genes including Pax1 and Pax9, we analyzed protein expression from these genes in these two cases compared to age‐matched controls. Immunochemical analysis showed a significant reduction in levels of Pax1 and Pax9 protein expression in chondrocytes of the vertebral column. Implications for the etiology and pathogenesis of JLS and related disorders are discussed.