Microtubule-mitochondrial attachment facilitates cell division symmetry and mitochondrial partitioning in fission yeast.

Association with microtubules inhibits the fission of mitochondria in Schizosaccharomyces pombe. Here, we show that this attachment of mitochondria to microtubules is an important cell-intrinsic factor in determining cell division symmetry. By comparing mutant cells that exhibited enhanced attachment and no attachment of mitochondria to microtubules (Dnm1Δ and Mmb1Δ, respectively), we show that microtubules in these mutants displayed aberrant dynamics compared to wild-type cells, which resulted in errors in nuclear positioning. This translated to cell division asymmetry in a significant proportion of both Dnm1Δ and Mmb1Δ cells. Asymmetric division in Dnm1Δ and Mmb1Δ cells resulted in unequal distribution of mitochondria, with the daughter cell that received more mitochondria growing faster than the other daughter cell. Taken together, we show the existence of homeostatic feedback controls between mitochondria and microtubules in fission yeast, which directly influence mitochondrial partitioning and, thereby, cell growth. This article has an associated First Person interview with the first author of the paper.

Mitochondrial dynamics, positioning and function mediated by cytoskeletal interactions.

The ability of a mitochondrion to undergo fission and fusion, and to be transported and localized within a cell are central not just to proper functioning of mitochondria, but also to that of the cell. The cytoskeletal filaments, namely microtubules, F-actin and intermediate filaments, have emerged as prime movers in these dynamic mitochondrial shape and position transitions. In this review, we explore the complex relationship between the cytoskeleton and the mitochondrion, by delving into: (i) how the cytoskeleton helps shape mitochondria via fission and fusion events, (ii) how the cytoskeleton facilitates the translocation and anchoring of mitochondria with the activity of motor proteins, and (iii) how these changes in form and position of mitochondria translate into functioning of the cell.

Cortical tethering of mitochondria by the anchor protein Mcp5 enables uniparental inheritance.

During sexual reproduction in eukaryotes, processes such as active degradation and dilution of paternal mitochondria ensure maternal mitochondrial inheritance. In the isogamous organism fission yeast, we employed high-resolution fluorescence microscopy to visualize mitochondrial inheritance during meiosis by differentially labeling mitochondria of the two parental cells. Remarkably, mitochondria, and thereby mitochondrial DNA from the parental cells, did not mix upon zygote formation but remained segregated at the poles by attaching to clusters of the anchor protein Mcp5 via its coiled-coil domain. We observed that this tethering of parental mitochondria to the poles results in uniparental inheritance of mitochondria, wherein two of the four spores formed subsequently contained mitochondria from one parent and the other spores contained mitochondria from the other parent. Further, the presence of dynein on an Mcp5 cluster precluded the attachment of mitochondria to the same cluster. Taken together, we reveal a distinct mechanism that achieves uniparental inheritance by segregation of parental mitochondria.

Association of mitochondria with microtubules inhibits mitochondrial fission by precluding assembly of the fission protein Dnm1.

Mitochondria are organized as tubular networks in the cell and undergo fission and fusion. Although several of the molecular players involved in mediating mitochondrial dynamics have been identified, the precise cellular cues that initiate mitochondrial fission or fusion remain largely unknown. In fission yeast (Schizosaccharomyces pombe), mitochondria are organized along microtubule bundles. Here, we employed deletions of kinesin-like proteins to perturb microtubule dynamics and used high-resolution and time-lapse fluorescence microscopy, revealing that mitochondrial lengths mimic microtubule lengths. Furthermore, we determined that compared with WT cells, mutant cells with long microtubules exhibit fewer mitochondria, and mutant cells with short microtubules have an increased number of mitochondria because of reduced mitochondrial fission in the former and elevated fission in the latter. Correspondingly, upon onset of closed mitosis in fission yeast, wherein interphase microtubules assemble to form the spindle within the nucleus, we observed increased mitochondrial fission. We found that the consequent rise in the mitochondrial copy number is necessary to reduce partitioning errors during independent segregation of mitochondria between daughter cells. We also discovered that the association of mitochondria with microtubules physically impedes the assembly of the fission protein Dnm1 around mitochondria, resulting in inhibition of mitochondrial fission. Taken together, we demonstrate a mechanism for the regulation of mitochondrial fission that is dictated by the interaction between mitochondria and the microtubule cytoskeleton.

Quantification of Mitochondrial Dynamics in Fission Yeast.

Mitochondria are double-membraned organelles responsible for several functions in the cell including energy production, calcium signaling, and cellular metabolism. An equilibrium between fission and fusion events of mitochondria is required for their proper functioning. Mitochondrial morphologies have been quantified in yeast using image processing modules such as MitoGraph and MitoLoc. However, the dynamics of mitochondrial fission and fusion have not been analyzed in these methods. Here, we present a method for measuring mitochondrial morphologies, as well as estimation of fission and fusion frequencies of mitochondria in individual fission yeast cells whose mitochondria are fluorescently-tagged or stained. The latter relies on counting of individual mitochondria upon signal filtering in each frame of a time-lapse. Taken together, we present a simple protocol for analyzing mitochondrial dynamics, which can easily be adopted to other model systems.

Role of Microtubules in Osteogenic Differentiation of Mesenchymal Stem Cells on 3D Nanofibrous Scaffolds.

Human bone marrow mesenchymal stem cells (MSCs) cultured on three-dimensional (3D) nanofibrous scaffolds are known to undergo osteogenic differentiation even in the absence of soluble osteoinductive factors. Although this process of differentiation has been attributed to the shape that cells assume on the fibrous scaffolds, it is unclear how constriction of cell shape would contribute to the differentiation phenotype. Here, we quantitatively compared cell and nuclear morphologies of cells cultured on 3D poly(ε-caprolactone) (PCL) nanofibers (NF) and two-dimensional (2D) flat films using confocal fluorescence microscopy. We discovered that while cells on the 2D films exhibited cellular and nuclear morphologies similar to those cultured on tissue culture polystyrene, cells cultured on the 3D NF showed distinct cell and nuclear morphologies, with lower areas and perimeters, but higher aspect ratios. We next tested the effect of treatment of cells with actin-depolymerizing cytochalasin D and microtubule-depolymerizing nocodazole on these morphologies. In both 2D and 3D scaffolds, actin depolymerization brought about gross changes in cell and nuclear morphologies. Remarkably, microtubule depolymerization resulted in a phenotype similar to actin depolymerization in cells cultured on 3D NF alone, indicating a significant role for the microtubule cytoskeleton in the maintenance of cell shape and structure in 3D. The morphological changes of the nucleus that were apparent upon cytoskeletal perturbation were reflected in the organization of heterochromatin in the nucleus, with MSCs on 3D alone exhibiting a differentiation phenotype. Finally, we tested the effect of cytoskeletal depolymerization on mineralization of cells. Again, we observed higher mineralization in cells cultured on 3D NF, which was lost in cells treated with either cytochalasin D or nocodazole. Taken together, our results suggest that both the actin and microtubule cytoskeletons contribute significantly toward maintenance of cell and nuclear shape in cells cultured on 3D scaffolds, and consequently to their osteogenic differentiation.