ABSTRACT
With the availability of increasing numbers of fluorescent protein variants and state-of-the-art imaging techniques, live cell microscopy has become a standard procedure in modern cell biology. Fluorescent markers are used to visualize the dynamic processes that take place in living cells, including the behavior of membrane-bound organelles. Here, we provide two examples of how we analyze the membrane dynamics of mitochondria in living yeast cells using wide field and confocal microscopy: (1) Long-term observation of mitochondrial shape changes using mitochondria-targeted fluorescent proteins and (2) monitoring the behavior of individual mitochondria using a mitochondria-targeted version of a photoconvertible fluorescent protein.
Subject(s)
Microscopy, Fluorescence , Mitochondrial Membranes/metabolism , Saccharomyces cerevisiae/metabolism , Gene Expression Regulation, Fungal , Luminescent Proteins/genetics , Luminescent Proteins/metabolism , Microscopy, Fluorescence/methods , Saccharomyces cerevisiae/cytology , Saccharomyces cerevisiae/geneticsABSTRACT
During the cell cycle each organelle has to be faithfully partitioned to the daughter cells. However, the mechanisms controlling organellar inheritance remain poorly understood. We studied the contribution of the cell cortex protein, Num1, to mitochondrial partitioning in yeast. Live-cell microscopy revealed that Num1 is required for attachment of mitochondria to the cell cortex and retention in mother cells. Electron tomography of anchoring sites revealed plasma membrane invaginations directly contacting the mitochondrial outer membrane. Expression of chimeric plasma membrane tethers rescued mitochondrial fission defects in Δnum1 and Δmdm36 mutants. These findings provide new insights into the coupling of mitochondrial dynamics, immobilization, and retention during inheritance.
Subject(s)
Cell Cycle/genetics , Cytoplasm/genetics , Cytoskeletal Proteins/genetics , Gene Expression Regulation, Fungal , Mitochondria/genetics , Mitochondrial Dynamics/genetics , Saccharomyces cerevisiae Proteins/genetics , Saccharomyces cerevisiae/genetics , Cytoplasm/metabolism , Cytoplasm/ultrastructure , Cytoskeletal Proteins/deficiency , Genes, Reporter , Green Fluorescent Proteins/genetics , Green Fluorescent Proteins/metabolism , Mitochondria/metabolism , Mitochondria/ultrastructure , Mitochondrial Membranes/metabolism , Mitochondrial Membranes/ultrastructure , Saccharomyces cerevisiae/metabolism , Saccharomyces cerevisiae/ultrastructure , Time-Lapse ImagingABSTRACT
The inheritance of mitochondria in yeast depends on bud-directed transport along actin filaments. It is a matter of debate whether anterograde mitochondrial movement is mediated by the myosin-related motor protein Myo2 or by motor-independent mechanisms. We show that mutations in the Myo2 cargo binding domain impair entry of mitochondria into the bud and are synthetically lethal with deletion of the YPT11 gene encoding a rab-type guanosine triphosphatase. Mitochondrial distribution defects and synthetic lethality were rescued by a mitochondria-specific Myo2 variant that carries a mitochondrial outer membrane anchor. Furthermore, immunoelectron microscopy revealed Myo2 on isolated mitochondria. Thus, Myo2 is an essential and direct mediator of bud-directed mitochondrial movement in yeast. Accumulating genetic evidence suggests that maintenance of mitochondrial morphology, Ypt11, and retention of mitochondria in the bud contribute to Myo2-dependent inheritance of mitochondria.
