Calum Thomson

Evidence for prebudding arrest of ER export in animal cell mitosis and its role in generating Golgi partitioning intermediates.

During mitosis the interconnected Golgi complex of animal cells breaks down to produce both finely dispersed elements and discrete vesiculotubular structures. The endoplasmic reticulum (ER) plays a controversial role in generating these partitioning intermediates and here we highlight the importance of mitotic ER export arrest in this process. We show that experimental inhibition of ER export (by microinjecting dominant negative Sar1 mutant proteins) is sufficient to induce and maintain transformation of Golgi cisternae to vesiculotubular remnants during interphase and telophase, respectively. We also show that buds on the ER, ER exit sites and COPII vesicles are markedly depleted in mitotic cells and COPII components Sec23p, Sec24p, Sec13p and Sec31p redistribute into the cytosol, indicating ER export is inhibited at an early stage. Finally, we find a markedly uneven distribution of Golgi residents over residual exit sites of metaphase cells, consistent with tubulovesicular Golgi remnants arising by fragmentation rather than redistribution via the ER. Together, these results suggest selective recycling of Golgi residents, combined with prebudding cessation of ER export, induces transformation of Golgi cisternae to vesiculotubular remnants in mitotic cells. The vesiculotubular Golgi remnants, containing populations of slow or nonrecycling Golgi components, arise by fragmentation of a depleted Golgi ribbon independently from the ER.

Carbonic anhydrase isoenzymes I, II, III, and IV are present in human esophageal epithelium.

Carbonic anhydrase (CA) isoenzymes have been widely studied in the gastrointestinal tract, where they mediate membrane transport events and pH regulation. However, the esophagus has generally received scant attention. In an immunohistochemical study confirmed by Western blotting, we have detected four CA isoenzymes (CAI, II, III, and IV) in the epithelium of human esophagus. Isoenzymes I, III, and sometimes IV (<10%) were present in the cytoplasm of basal cells and II and IV in the cytoplasm and cell surface membranes, respectively, of suprabasal cells (prickle cells). The localization of CAIV to the plasma membranes was confirmed by electron microscopic immunocytochemistry. CA was effectively divided at the basal-suprabasal interface between low-activity CAI and III (basal) and high-activity CAII and IV (suprabasal). Carbonic anhydrase in esophageal epithelial cells may have several functions: elimination of CO2 and metabolites, participation in membrane transport events during active cell growth, and pH regulation as a protective mechanism against acidic gastric reflux.

mito-QC illuminates mitophagy and mitochondrial architecture in vivo

Whether mitophagy occurs within specific cellular subtypes in vivo is unclear. McWilliams et al. present “mito-QC,” a transgenic mouse containing a pH-sensitive fluorescent mitochondrial signal, allowing in vivo detection of mitophagy and mitochondrial morphology at single-cell resolution.

Keratin 9 Is Required for the Structural Integrity and Terminal Differentiation of the Palmoplantar Epidermis

Keratin 9 (K9) is a type I intermediate filament protein whose expression is confined to the suprabasal layers of the palmoplantar epidermis. Although mutations in the K9 gene are known to cause epidermolytic palmoplantar keratoderma, a rare dominant-negative skin disorder, its functional significance is poorly understood. To gain insight into the physical requirement and importance of K9, we generated K9-deficient (Krt9−/−) mice. Here, we report that adult Krt9−/−mice develop calluses marked by hyperpigmentation that are exclusively localized to the stress-bearing footpads. Histological, immunohistochemical, and immunoblot analyses of these regions revealed hyperproliferation, impaired terminal differentiation, and abnormal expression of keratins K5, K14, and K2. Furthermore, the absence of K9 induces the stress-activated keratins K6 and K16. Importantly, mice heterozygous for the K9-null allele (Krt9+/−) show neither an overt nor histological phenotype, demonstrating that one Krt9 allele is sufficient for the developing normal palmoplantar epidermis. Together, our data demonstrate that complete ablation of K9 is not tolerable in vivo and that K9 is required for terminal differentiation and maintaining the mechanical integrity of palmoplantar epidermis.

A comparison of membrane enzymes of human and pig oesophagus; the pig oesophagus is a good model for studies of the gullet in man

SummaryThe distribution and relative catalytic activities of five plasma membrane enzymes (alkaline phosphatase, dipeptidyl peptidase IV, γ-glutamyl transpeptidase, microsomal alanyl aminopeptidase and glutamyl aminopeptidase) were examined in human and pig oesophagus. In both species, alkaline phosphatase activity occurred in basal and suprabasal cells of the epithelium and in capillaries. Stromal cells in the human submucosa were particularly reactive. Dipeptidyl peptidase IV was present in blood vessels and capillaries in man and pig and in submucous glands in the pig. The enzyme was also present in both species in the lamina propria cells immediately adjacent to the epithelial basal lamina. In the human, γ-glutamyl transpeptidase occurred in the epithelial basal cells and in isolated basal and lower prickle cells in the pig. Stromal cells in the human submucosa were strongly reactive and capillaries in the muscularis propria in both species moderately active. Microsomal alanyl aminopeptidase was detected in lamina propria cells adjacent to the epithelial basal cell layer in man and pig and at the apices of mucous cells in pig submucous glands. Weak glutamyl aminopeptidase activity was confined to capillaries in both species. The findings of this study, along with the ready availability of pig oesophagus, suggest that the pig may be a suitable model for studies of the gullet in man.

Carbonic anhydrase is present in human oesophageal epithelium and submucosal glands

SummaryCarbonic anhydrase (EC 4.2.1.1) activity was investigated in normal human oesophageal mucosa using the Hansson and Ridderstråle catalytic cobalt methods. The enzyme was detected in the cell membranes and nuclei and, to a lesser extent, in the cytoplasm of the epithelial cells of the mucosa giving a ‘chicken wire’ appearance. Activity decreased towards the lumen. Other stratified squamous epithelia - buccal mucosa, ectocervix and skin - gave a similar pattern. Acinar cells of oesophageal submucosal glands also exhibited activity for the enzyme, but the ducts did not. The formation of reaction product was prevented by acetazolamide and ethoxzolamide and by the omission of bicarbonate from the substrate medium. Carbonic anhydrase in oesophageal squamous epithelium may be involved in the control of intra- and extracellular pH, while that in the glands is more likely to be concerned with bicarbonate secretion.

The distribution of carbonic anhydrase II in human, pig and rat oesophageal epithelium.

Carbonic anhydrase II (CA II) is present in human oesophageal epithelial cells and probably involved in protecting the mucosa against acidic gastric refluxate. If this is the case, then it is likely that the enzyme will be more concentrated at or near the gastro-oesophageal junction. To answer this question, and determine whether CA II is present and similarly distributed in other species, we also examined the oesophageal epithelium of the rat and pig. In the rat, CA II was largely absent from the oesophageal epithelium, but present in the stratified squamous epithelium of the gastric forestomach as an approximately 2 mm-long collar around the entrance to the corpus, a site that roughly corresponds to the gastro-oesophageal junction in other animals. The enzyme was present mainly in basal and prickle cells. In upper and middle pig oesophagus, CA II was largely confined to basal cells and isolated groups of stratified superficial prickle cells. CA II-containing epithelial cells were highly concentrated in the thickened epithelium at the gastro-oesophageal junction (about four-times thicker than upper or middle). Reactive cells were present throughout the depth of the epithelium, but noticeably more concentrated in the basal and superficial prickle cell layers. CA II was also prominent in the most superficial cell layers in islands of the oesophageal mucosa within the gastric cardia. In man, CA II was confined largely to the basal half of the epithelium in the upper and middle regions of oesophagus. The distribution of CA II at the gastro-oesophageal junction took different forms. In general, there were more CA II-reactive cells at or closer to the lumen. The superficial prickle cell layers tended to exhibit more CA II than the deeper layers, with basal and epibasal cells containing little or no enzyme. In other regions of the same specimens, CA II-containing cells were present from the basal to the most luminal layers. If CA II in oesophageal epithelial cells in the region of the gastro-oesophageal junction (or in the case of the rat the forestomach/corpus junction) is important in the defence against refluxate, then it is in a vulnerable site, since bile salts are potent inhibitors of the enzyme. The action of bile salts on CA II may be an important factor in the initiation of oesophageal disease.

A HISTOCHEMICAL STUDY OF CARBONIC ANHYDRASE IN THE PLASMA MEMBRANES OF HUMAN ORAL EPITHELIAL CELLS

Carbonic anhydrase (EC 4.2.1.1) was detected histochemically from the following regions in patients of various ages (14-84 yr): buccal mucosa, buccal flap, hard palate and tongue. The enzyme was principally located in the cell membranes but was also present in nuclei. There was a gradation in activity from basal (strong) to superficial cells (weak/negative). The carbonic anhydrase inhibitors ethoxyzolamide and acetazolamide abolished activity at 0.001 mM, but were ineffective, even at 1.2 mM, against a reaction associated with the granules of the stratum granulosum. No activity was detected in the absence of bicarbonate from the substrate.

Targets of Wnt/ß-catenin transcription in penile carcinoma.

Penile squamous cell carcinoma (PeCa) is a rare malignancy and little is known regarding the molecular mechanisms involved in carcinogenesis of PeCa. The Wnt signaling pathway, with the transcription activator ß-catenin as a major transducer, is a key cellular pathway during development and in disease, particularly cancer. We have used PeCa tissue arrays and multi-fluorophore labelled, quantitative, immunohistochemistry to interrogate the expression of WNT4, a Wnt ligand, and three targets of Wnt-ß-catenin transcription activation, namely, MMP7, cyclinD1 (CD1) and c-MYC in 141 penile tissue cores from 101 unique samples. The expression of all Wnt signaling proteins tested was increased by 1.6 to 3 fold in PeCa samples compared to control tissue (normal or cancer adjacent) samples (p<0.01). Expression of all proteins, except CD1, showed a significant decrease in grade II compared to grade I tumors. High magnification, deconvolved confocal images were used to measure differences in co-localization between the four proteins. Significant (p<0.04-0.0001) differences were observed for various permutations of the combinations of proteins and state of the tissue (control, tumor grades I and II). Wnt signaling may play an important role in PeCa and proteins of the Wnt signaling network could be useful targets for diagnosis and prognostic stratification of disease.

An immunocytochemical study of the carbonic anhydrase I isoenzyme in human oral Merkel cells.

Merkel cells in human buccal mucosa and hard palate possess the carbonic anhydrase I isoenzyme (CAI). CAI colocalized immunocytochemically with a range of Merkel cell cytokeratins, namely CK 7, 8, 18, 19 and 20. No other cells in the oral epithelium were immunoreactive for the CAI antibody. The presence of the enzyme may be related to the function of sensory receptors that produce a sustained response to a maintained mechanical stimulus.