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Combined Atomic Force Microscopy - Fluorescence Microscopy: Analyzing Exocytosis at Alveolar Cells.
MedLine Citation:
PMID:  22694258     Owner:  NLM     Status:  Publisher    
Hybrid atomic force microscopy (AFM) - fluorescence microscopy (FM) investigation of exocytosis at lung epithelial cells (ATII cells) allows the detection of individual exocytic events by FM, which can be simultaneously correlated to structural changes in individual cells by AFM. Exocytosis of lamellar bodies (LBs) represents a slow form of exocytosis found in many non-neuronal cells. Exocytosis of LBs, following stimulation with Adenosine-5`-triphosphate (ATP) and Phorbol 12-myristate 13-acetate (PMA) results in a cation influx via P2X4 receptors at the site of LB fusion with the plasma membrane (PM), which should induce a temporary increase of cell height/volume. AFM measurements were performed in single-line scans across the cell surface. Five minutes after stimulation, ATII cells revealed a cell height and volume increase of 13.7 % ± 4.1 % and 15.9 ± 4.8 % (N=9), respectively. These transient changes depend on exocytic LB - PM fusion. Non-stimulated cells and cells lacking LB fusions did not show a significant change in cell height/volume (N=8). In addition, a cell height decrease was observed in ATII cells stimulated by Uridine-5`-triphosphate (UTP) and PMA, agonists inducing LB fusion with the PM, but not activation of P2X4 receptors. The cell height and volume decreased by -8.0 ± 4.5 % and -10.8 ± 5.0 % (N=3), respectively. Additionally, low force contact and dynamic mode AFM imaging of cell areas around the nucleus after stimulation with ATP/PMA was performed. Fused LBs are more pronounced in AFM topography images compared to non-fused LBs concluding that different 'dynamic states' of LBs or locations from the PM are captured during imaging.
Elena Hecht; Kristin Thompson; Manfred Frick; Oliver H Wittekindt; Paul Dietl; Boris Mizaikoff; Christine Kranz
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Publication Detail:
Type:  JOURNAL ARTICLE     Date:  2012-6-12
Journal Detail:
Title:  Analytical chemistry     Volume:  -     ISSN:  1520-6882     ISO Abbreviation:  -     Publication Date:  2012 Jun 
Date Detail:
Created Date:  2012-6-14     Completed Date:  -     Revised Date:  -    
Medline Journal Info:
Nlm Unique ID:  0370536     Medline TA:  Anal Chem     Country:  -    
Other Details:
Languages:  ENG     Pagination:  -     Citation Subset:  -    
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