Quantification of Local Morphodynamics and Local GTPase Activity by Edge Evolution Tracking

Advances in time-lapse fluorescence microscopy have enabled us to directly observe dynamic cellular phenomena. Although the techniques themselves have promoted the understanding of dynamic cellular functions, the vast number of images acquired has generated a need for automated processing tools to extract statistical information. A problem underlying the analysis of time-lapse cell images is the lack of rigorous methods to extract morphodynamic properties. Here, we propose an algorithm called edge evolution tracking (EET) to quantify the relationship between local morphological changes and local fluorescence intensities around a cell edge using time-lapse microscopy images. This algorithm enables us to trace the local edge extension and contraction by defining subdivided edges and their corresponding positions in successive frames. Thus, this algorithm enables the investigation of cross-correlations between local morphological changes and local intensity of fluorescent signals by considering the time shifts. By applying EET to fluorescence resonance energy transfer images of the Rho-family GTPases Rac1, Cdc42, and RhoA, we examined the cross-correlation between the local area difference and GTPase activity. The calculated correlations changed with time-shifts as expected, but surprisingly, the peak of the correlation coefficients appeared with a 6–8 min time shift of morphological changes and preceded the Rac1 or Cdc42 activities. Our method enables the quantification of the dynamics of local morphological change and local protein activity and statistical investigation of the relationship between them by considering time shifts in the relationship. Thus, this algorithm extends the value of time-lapse imaging data to better understand dynamics of cellular function

Recurrent pneumonia with mild hypogammaglobulinemia diagnosed as X-linked agammaglobulinemia in adults

<p>Abstract</p> <p>Background</p> <p>X-linked agammaglobulinemia (XLA) is a humoral immunodeficiency caused by disruption of the Bruton's tyrosine kinase (BTK) gene. Typical XLA patients suffer recurrent and severe bacterial infections in childhood.</p> <p>Methods</p> <p>Flow cytometric analysis of the peripheral monocytes using the anti-BTK antibody was used to characterize a 27 year old male patient with mild hypogammaglobulinemia (IgG, 635 mg/dl; IgM, 11 mg/dl; IgA, <5 mg/dl). He had suffered from frequent pneumonia since age 25 but had no history of frequent infections in his childhood or in adolescence. Sequencing of the BTK cDNA obtained from an Epstein–Barr virus-transformed B lymphoblastoid cell line derived from the bone marrow of the patient was performed to confirm a genetic defect.</p> <p>Results</p> <p>Flow cytometric analysis of cytoplasmic BTK protein in peripheral monocytes indicated that the patient presents a rare case of adult-onset XLA and that his mother is an XLA carrier. Sequencing of the BTK gene revealed a deletion of AG in the codon for Glu605 (AGT), resulting in an aberrant stop codon that truncates the BTK protein in its kinase domain.</p> <p>Conclusions</p> <p>This case suggests that some XLA cases may remain undiagnosed because they only show mild hypogammaglobulinemia and they lack repeated infections in childhood. Flow cytometric analysis is a powerful method to screen these patients.</p

Up-regulation of Na+,K+-ATPase α3-isoform and down-regulation of the α1-isoform in human colorectal cancer

AbstractWe investigated expression levels of Na+,K+-ATPase α-isoforms and their ATPase activities in human colorectal cancer tissue and the accompanying normal mucosa. A decrease in expression of the α1-isoform protein was observed in all sampled cancer tissues compared with the normal mucosae. The level of ouabain (5 μM)-sensitive Na+,K+-ATPase activity in carcinomas was 81±5% that of in the normal mucosae. The mRNA expression of α2- and α4-isoforms was decreased in almost all the carcinoma samples. Interestingly, the expression level of the α3-isoform protein in the cancer tissue was higher than that of the normal mucosa. These results indicate that a decrease in the α1-isoform expression and an increase in the α3-isoform expression may be associated with colorectal cancer

K+-Cl- Cotransporter-3a Up-regulates Na+,K+-ATPase in Lipid Rafts of Gastric Luminal Parietal Cells*

Gastric parietal cells migrate from the luminal to the basal region of the gland and they gradually lose acid secretory activity. So far, distribution and function of K+-Clcotransporters (KCCs) in gastric parietal cells have not been reported. We found that KCC3a but not KCC3b mRNA was highly expressed and KCC3a protein was predominantly expressed in the basolateral membrane of rat gastric parietal cells located in the luminal region of the glands. KCC3a and Na+,K+-ATPase α1-subunit (α1NaK) were co-immunoprecipitated and both of them were highly localized in a lipid raft fraction. The ouabain-sensitive K+-dependent ATP-hydrolyzing activity (Na+,K+-ATPase activity) was significantly inhibited by a KCC inhibitor (DIOA). The stable exogenous expression of KCC3a in LLC-PK1 cells resulted in association of KCC3a with endogenous α1NaK and it recruited α1NaK in lipid rafts, accompanying increases of Na+,K+-ATPase activity and ouabain-sensitive Na+ transport activity which were suppressed by DIOA, while the total expression level of α1NaK in the cells was not significantly altered. On the other hand, the expression of KCC4 induced no association with α1NaK. In conclusion, KCC3a forms a functional complex with α1NaK in the basolateral membrane of luminal parietal cells and it up-regulates α1NaK in lipid rafts, while KCC3a is absent in basal parietal cells

Mineral paragenesis and its implications in the högbomite-bearing skarn, Sør Rondane Mountains, East Antarctica

第2回極域科学シンポジウム/第31回極域地学シンポジウム 11月17日（木） 国立極地研究所　2階大会議