Abstract. Using fluorescent membrane potential sensing dyes to stain budding yeast, mitochondria are resolved as tubular organeUes aligned in radial arrays that converge at the bud neck. Time-lapse fluorescence microscopy reveals region-specific, directed mitochondrial movement during polarized yeast cell growth and mitotic cell division. Mitochondria in the central region of the mother cell move linearly towards the bud, traverse the bud neck, and progress towards the bud tip at an average velocity of 49 _ 21 nm/sec. In contrast, mitochondria in the peripheral region of the mother cell and at the bud tip display significantly less movement. Yeast strains containing temperature sensitive lethal mutations in the actin gene show abnormal mitochondrial distribution. No mitochondrial movement is evident in these mutants after short-term shift to semipermissive temperatures. Thus, the actin cytoskeleton is important for normal mitochondrial movement during inheritance. To determine the possible role of known myosin genes in yeast mitochondrial motility, we investigated mitochondrial inheritance in myol, rnyo2, myo3 and myo4 single mutants and in a myo2, myo4 double mutant. Mitochondrial spatial arrangement and motility are not significantly affected by these mutations. We used a microfilament sliding assay to examine motor activity on isolated yeast mitochondria. Rhodamine-phalloidin labeled yeast actin filaments bind to immobilized yeast mitochondria, as well as unilamellar, right-side-out, sealed mitochondrial outer membrane vesicles. In the presence of low levels of ATP (0.1-100 ~M), we observe F-actin sliding on immobilized yeast mitochondria. In the presence of high levels of ATP (500 ~M-2 mM), bound filaments are released from mitochondria and mitochondrial outer membranes. The maximum velocity of mitochondriadriven microfilament sliding (23 + 11 nm/sec) is similar to that of mitochondrial movement in living cells. This motor activity requires hydrolysis of ATP, does not require cytosolic extracts, is sensitive to protease treatment, and displays an ATP concentration dependence similar to that of members of the myosin family of actin-based motors. This is the first demonstration of an actin-based motor activity in a defined organelle population. MITOCHONDRIAL inheritance is the process whereby mitochondria are transferred from mother cells to developing daughter cells during cell division.In the budding yeast, Saccharomyces cerevisiae, this process begins at the G1/S boundary of the cell division cycle (Stevens, 1981). Since mitochondria are produced only from preexisting mitochondria (Criddle and Schatz, 1969;Schatz and Saltzgaber, 1969), this inheritance process ensures that each daughter cell contains this indispensable organelle. We used budding yeast to study the dynamics of mitochondrial movements leading to inheritance, and the organelle-cytoskeletal interactions that control these movements.Cell division in S. cerevisiae proceeds by asymmetric growth of the developing daughter cell. In yeast and otherPl...

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During early stages of meiosis I, yeast mitochondria fuse to form a single continuous thread. Thereafter, portions of the mitochondrial thread are equally distributed to daughter cells. Using time-lapse fluorescence microscopy and a membrane potential sensing dye, mitochondria are resolved as small particles at the cell periphery in pre-meiotic, living yeast. These organelles display low levels of movement. During meiosis I, we observed a threefold increase in mitochondrial motility. Mitochondrial movements were linear, occurred at a maximum velocity of 25 +/- 6.7 nm/s, and resulted in organelle collision and fusion to form elongated tubular structures. Mitochondria do not co-localize with microtubules. Destabilization of microtubules by nocodazole treatment has no significant effect on the rate and extent of thread formation. In contrast, yeast bearing temperature-sensitive mutations in the actin-encoding ACT1 gene (act1-3 and act1-133) exhibit abnormal mitochondrial aggregation, fragmentation, and enlargement as well as loss of mitochondrial motility. In act1-3 cells, mitochondrial defects and actin delocalization occur only at restrictive temperatures. The act1-133 mutation, which perturbs the myosin-binding site of actin without significantly affecting actin cytoskeletal structure in meiotic yeast, results in mitochondrial morphology and motility defects at restrictive and permissive temperatures. These studies support a role for the actin cytoskeleton in the control of mitochondrial position and movements in meiotic yeast.

Changes in intracellular Ca2+ correlate with specific events in the cell cycle. Here we investigated the role of Ca2+ in the G1 phase. HEK 293 cells were arrested in mitosis and subjected to short-term treatments that alter Ca2+ homeostasis prior to their release into G1. Treatment with thapsigargin (TG), an irreversible inhibitor of the sarco-endoplasmic reticulum Ca2+ ATPase (SERCA) lengthened the G1 phase. Moreover, TG treatment also resulted in a dramatic alteration in cellular morphology and attachment and in the reduction of MAPK activity and lower levels of cyclin D1 and cyclin E proteins. Treatments with reagents that transiently increase or decrease cytosolic Ca2+ or that temporarily inactivate SERCA did not alter any of the above parameters. Cells expressing a TG-resistant form of SERCA progressed normally through the G1/S transition after TG treatment. These results suggest that long-term SERCA inactivation affects cell cycle-dependent events and compromises progression through G1/S.

Evidence for actin-dependent organelle movement was first obtained from studies of cytoplasmic streaming in plants. These studies, together with cell-free organelle motility studies and biophysical analyses of muscle myosin, support a model whereby organelle-associated motor molecules utilize the energy of adenosine triphosphate binding and hydrolysis to drive movement along F-actin tracks. Recent studies indicate that this mechanism for organelle movement may be responsible for organelle and vesicle movement during secretion, endocytosis and mitochondrial inheritance in a variety of eukaryotes.