4Pi microscopy reveals an impaired three-dimensional mitochondrial network of pancreatic islet beta-cells, an experimental model of type-2 diabetes.
Insulin production in pancreatic beta-cells is critically linked to mitochondrial oxidative phosphorylation. Increased ATP production triggered by blood glucose represents the beta-cells' glucose sensor. Type-2 diabetes mellitus results from insulin resistance in peripheral tissues and impaired insulin secretion. Pathology of diabetic beta-cells might be reflected by the altered morphology of mitochondrial network. Its characterization is however hampered by the complexity and density of the three-dimensional (3D) mitochondrial tubular networks in these cell types. Conventional confocal microscopy does not provide sufficient axial resolution to reveal the required details; electron tomography reconstruction of these dense networks is still difficult and time consuming. However, mitochondrial network morphology in fixed cells can also be studied by 4Pi microscopy, a laser scanning microscopy technique which provides an approximately 7-fold improved axial resolution (approximately 100 nm) over conventional confocal microscopy. Here we present a quantitative study of these networks in insulinoma INS-1E cells and primary beta-cells in Langerhans islets. The former were a stably-transfected cell line while the latter were transfected with lentivirus, both expressing mitochondrial matrix targeted redox-sensitive GFP. The mitochondrial networks and their partial disintegration and fragmentation are revealed by carefully created iso-surface plots and their quantitative analysis. We demonstrate that beta-cells within the Langerhans islets from diabetic Goto Kakizaki rats exhibited a more disintegrated mitochondrial network compared to those from control Wistar rats and model insulinoma INS-1E cells. Standardization of these patterns may lead to development of morphological diagnostics for Langerhans islets, for the assessment of beta-cell condition, before their transplantations.
Dlasková
Andrea
A
Department of Membrane Transport Biophysics, No. 75, Institute of Physiology, Academy of Sciences of the Czech Republic, Prague, Czech Republic.
Spacek
Tomás
T
Santorová
Jitka
J
Plecitá-Hlavatá
Lydie
L
Berková
Zuzana
Z
Saudek
Frantisek
F
Lessard
Mark
M
Bewersdorf
Joerg
J
Jezek
Petr
P
D000818:Animals; D045744:Cell Line, Tumor; D003924:Diabetes Mellitus, Type 2 / Q000473:pathology*; D004195:Disease Models, Animal; D049452:Green Fluorescent Proteins / Q000235:genetics; D021621:Imaging, Three-Dimensional; D066298:In Vitro Techniques; D050417:Insulin-Secreting Cells / Q000473:pathology* / Q000648:ultrastructure; D007340:Insulinoma / Q000473:pathology; D018613:Microscopy, Confocal / Q000379:methods*; D008928:Mitochondria / Q000473:pathology* / Q000648:ultrastructure; D010190:Pancreatic Neoplasms / Q000473:pathology; D051381:Rats; D017208:Rats, Wistar; D011993:Recombinant Fusion Proteins / Q000235:genetics; D014162:Transfection
D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins