High-resolution imaging of mitochondria and mitochondrial nucleoids in differentiated SH-SY5Y cells

Mitochondria are highly dynamic organelles which form intricate networks with complex dynamics. Mitochondrial transport and distribution are essential to ensure proper cell function, especially in cells with an extremely polarised morphology such as neurons. A layer of complexity is added when considering mitochondria have their own genome, packaged into nucleoids. Major mitochondrial morphological transitions, for example mitochondrial division, often occur in conjunction with mitochondrial DNA (mtDNA) replication and changes in the dynamic behaviour of the nucleoids. However, the relationship between mtDNA dynamics and mitochondrial motility in the processes of neurons has been largely overlooked. In this chapter, we describe a method for live imaging of mitochondria and nucleoids in differentiated SH-SY5Y cells by instant structured illumination microscopy (iSIM). We also include a detailed protocol for the differentiation of SH-SY5Y cells into cells with a pronounced neuronal-like morphology and show examples of coordinated mitochondrial and nucleoid motility in the long processes of these cells.

Casein kinase TbCK1.2 regulates division of kinetoplast DNA, and movement of basal bodies in the African trypanosome

The single mitochondrial nucleoid (kinetoplast) of Trypanosoma brucei is found proximal to a basal body (mature (mBB)/probasal body (pBB) pair). Kinetoplast inheritance requires synthesis of, and scission of kinetoplast DNA (kDNA) generating two kinetoplasts that segregate with basal bodies into daughter cells. Molecular details of kinetoplast scission and the extent to which basal body separation influences the process are unavailable. To address this topic, we followed basal body movements in bloodstream trypanosomes following depletion of protein kinase TbCK1.2 which promotes kinetoplast division. In control cells we found that pBBs are positioned 0.4 um from mBBs in G1, and they mature after separating from mBBs by at least 0.8 um: mBB separation reaches ~2.2 um. These data indicate that current models of basal body biogenesis in which pBBs mature in close proximity to mBBs may need to be revisited. Knockdown of TbCK1.2 produced trypanosomes containing one kinetoplast and two nuclei (1K2N), increased the percentage of cells with uncleaved kDNA 400%, decreased mBB spacing by 15%, and inhibited cytokinesis 300%. We conclude that (a) separation of mBBs beyond a threshold of 1.8 um correlates with division of kDNA, and (b) TbCK1.2 regulates kDNA scission. We propose a Kinetoplast Division Factor hypothesis that integrates these data into a pathway for biogenesis of two daughter mitochondrial nucleoids.

Mitochondrial HMG-Box Containing Proteins: From Biochemical Properties to the Roles in Human Diseases

Mitochondrial DNA (mtDNA) molecules are packaged into compact nucleo-protein structures called mitochondrial nucleoids (mt-nucleoids). Their compaction is mediated in part by high-mobility group (HMG)-box containing proteins (mtHMG proteins), whose additional roles include the protection of mtDNA against damage, the regulation of gene expression and the segregation of mtDNA into daughter organelles. The molecular mechanisms underlying these functions have been identified through extensive biochemical, genetic, and structural studies, particularly on yeast (Abf2) and mammalian mitochondrial transcription factor A (TFAM) mtHMG proteins. The aim of this paper is to provide a comprehensive overview of the biochemical properties of mtHMG proteins, the structural basis of their interaction with DNA, their roles in various mtDNA transactions, and the evolutionary trajectories leading to their rapid diversification. We also describe how defects in the maintenance of mtDNA in cells with dysfunctional mtHMG proteins lead to different pathologies at the cellular and organismal level.