Complex patterns of mitochondrial dynamics in human pancreatic cells revealed by fluorescent confocal imaging

Abstract

Mitochondrial morphology and intracellular organization are tightly controlled by the processes of mitochondrial fission-fusion. Moreover, mitochondrial movement and redistribution provide a local ATP supply at cellular sites of particular demands. Here we analysed mitochondrial dynamics in isolated primary human pancreatic cells. Using real time confocal microscopy and mitochondria-specific fluorescent probes tetramethylrhodamine methyl ester and MitoTracker Green we documented complex and novel patterns of spatial and temporal organization of mitochondria, mitochondrial morphology and motility. The most commonly observed types of mitochondrial dynamics were (i) fast fission and fusion; (ii) small oscillating movements of the mitochondrial network; (iii) larger movements, including filament extension, retraction, fast (0.1-0.3 mum/sec.) and frequent oscillating (back and forth) branching in the mitochondrial network; (iv) as well as combinations of these actions and (v) long-distance intracellular translocation of single spherical mitochondria or separated mitochondrial filaments with velocity up to 0.5 mum/sec. Moreover, we show here for the first time, a formation of unusual mitochondrial shapes like rings, loops, and astonishingly even knots created from one or more mitochondrial filaments. These data demonstrate the presence of extensive heterogeneity in mitochondrial morphology and dynamics in living cells under primary culture conditions. In summary, this study reports new patterns of morphological changes and dynamic motion of mitochondria in human pancreatic cells, suggesting an important role of integrations of mitochondria with other intracellular structures and systems.

Fig 1

TMRM fluorescence in human pancreatic cells over time shows a slight decline and no oscillations of the signal intensity. Note that time of this test (∼2000 sec.) exceeds markedly a typical observation time used in our study. Data of five different cells are presented.

Fig 2

Typical mitochondrial network in human pancreatic cells visualized by mitochondrial fluorescent probe TMRM. In some cells the mitochondrial network covers the whole cell (left panel, arrows), whereas other cells show elongated mitochondrial threads surrounding nuclei (N). Note that mitochondrial morphology vary from small, sphere like mitochondria to very long up to 30 μm long mitochondrial filaments (right panel). Scale bar, 10 μm.

Fig 3

Fragmentation of mitochondrial network filaments in human pancreatic cells. (A) Continuous mitochondrial network. (B) Partially fragmented mitochondrial network with shorter separated filaments (dashed box). (C) Dynamics of mitochondrial fission. Fast division of long filament into three fragments (arrows) occurred during about 100 sec. (A) and (B): Scale bar, 5 μm. (C): Scale bar, 1 μm. The video file for Fig. 2C is available under ‘Supporting Information’.

Fig 4

Remodelling of mitochondrial network in human pancreatic cells. Dynamics of network fragmentation (A) and fusion of two network branches (B) is shown (arrows). Note that similar time (∼100 sec.) is required for both fission and fusion processes (cf. Fig. 2C). Scale bar, 1 μm.

Fig 5

Mitochondrial dynamic motion in human pancreatic cells. Rapid intracellular displacement (move and stop) of individual mitochondrion (arrow) with velocity of ∼0.25 μm/sec. can be seen. Note that another similar size and shape mitochondrion (asterisk) is fixed in its position. Scale bar, 1 μm. The video file for this figure is available under ‘Supporting Information’.

Fig 6

Complex pattern of mitochondrial motility in human pancreatic cells. Combination of (i) small amplitude fluctuations, (ii) long-distance (∼20 μm) occasional translocation of single mitochondrion (circles, dotted arrows) and (iii) fast ‘back and forth’ branching in mitochondrial network (arrows) is shown. Scale bar, 5 μm. The video file for this figure is available under ‘Supporting Information’.

Fig 7

Dynamic ring formation at the end of mitochondrial filament. Note that this process can be reversible (dashed circle). Scale bar, 1 μm.

Fig 8

Combination of ring at the end of mitochondrial filament with fast branch formation. Note that more intense signal is observed in the sites of mitochondrial branch formation (dashed circle). Scale bar, 1 μm.

Fig 9

New level of the complexity of mitochondrial dynamics in human pancreatic cells. Dynamic formation of mitochondrial ‘knot’ from single mitochondrial filament. Complex multidirectional movement of mitochondrial thread is indicated by arrows. Scale bar, 1 μm. The video file for this figure is available under ‘Supporting Information’.

Fig 10

Formation of mitochondrial dynamic ‘knot’ from separated mitochondrial threads (arrow). Scale bar, 1 μm. The video file for this figure is available under ‘Supporting Information’.