Mitochondrial fusion remains a largely unknown process despite its observation by live microscopy and the identification of few implicated proteins. Using green and red fluorescent proteins targeted to the mitochondrial matrix, we show that mitochondrial fusion in human cells is efficient and achieves complete mixing of matrix contents within 12 h. This process is maintained in the absence of a functional respiratory chain, despite disruption of microtubules or after significant reduction of cellular ATP levels. In contrast, mitochondrial fusion is completely inhibited by protonophores that dissipate the inner membrane potential. This inhibition, which results in rapid fragmentation of mitochondrial filaments, is reversible: small and punctate mitochondria fuse to reform elongated and interconnected ones upon withdrawal of protonophores. Expression of wild-type or dominant-negative dynamin-related protein 1 showed that fragmentation is due to dynamin-related protein 1-mediated mitochondrial division. On the other hand, expression of mitofusin 1 (Mfn1), one of the human Fzo homologues, increased mitochondrial length and interconnectivity. This process, but not Mfn1 targeting, was dependent on the inner membrane potential, indicating that overexpressed Mfn1 stimulates fusion. These results show that human mitochondria represent a single cellular compartment whose exchanges and interconnectivity are dynamically regulated by the balance between continuous fusion and fission reactions.

/pdf/mitochondrial-fusion-in-human-cells-is-efficient-requires-mj6o5o1ove.pdf

Mitochondrial fusion in human cells is efficient, requires the inner membrane potential, and is mediated by mitofusins.

A critical physiological role of zinc in the structure and function of biomembranes.

The nuclear envelope links the cytoskeleton to structural components of the nucleus. It functions to coordinate nuclear migration and anchorage, organize chromatin, and aid meiotic chromosome pairing. Forces generated by the cytoskeleton are transferred across the nuclear envelope to the nuclear lamina through a nuclear-envelope bridge consisting of SUN (Sad1 and UNC-84) and KASH (Klarsicht, ANC-1 and Syne/Nesprin homology) proteins. Some KASH-SUN combinations connect microtubules, centrosomes, actin filaments, or intermediate filaments to the surface of the nucleus. Other combinations are used in cell cycle control, nuclear import, or apoptosis. Interactions between the cytoskeleton and the nucleus also affect global cytoskeleton organization. SUN and KASH proteins were identified through genetic screens for mispositioned nuclei in model organisms. Knockouts of SUN or KASH proteins disrupt neurological and muscular development in mice. Defects in SUN and KASH proteins have been linked to human diseases including muscular dystrophy, ataxia, progeria, lissencephaly, and cancer.

/pdf/interactions-between-nuclei-and-the-cytoskeleton-are-2a1cazipdt.pdf

Interactions Between Nuclei and the Cytoskeleton Are Mediated by SUN-KASH Nuclear-Envelope Bridges

We have generated several stable cell lines expressing GFP-labeled centrin. This fusion protein becomes concentrated in the lumen of both centrioles, making them clearly visible in the living cell. Time-lapse fluorescence microscopy reveals that the centriole pair inherited after mitosis splits

/pdf/the-respective-contributions-of-the-mother-and-daughter-46hukem5xa.pdf

The respective contributions of the mother and daughter centrioles to centrosome activity and behavior in vertebrate cells.

The C. elegans Hook Protein, ZYG-12, Mediates the Essential Attachment between the Centrosome and Nucleus

THE persistence of the centriole during interphase near the nucleus within the so-called cell centre seems to be a general feature of most animal cells. An association between the nucleus and the cell centre has been proposed and has been clearly shown in some cell types1,2. A permanent system involving chromosomes, spindle fibres and centrioles has been suggested by Lettre et al.3, and Aronson4 has demonstrated the existence of a Colcemid-sensitive mobile system between the nucleus and a centre at the two-cell stage in the sea urchin Lytechinus variegatus. We show here that there is a physical association between the centriole and the nucleus in the rat liver cell which resists cold treatment, cell disruption and nuclear isolation. When nuclear envelopes are prepared from isolated nuclei, the association between the centriole and the nuclear envelope is maintained.

Is the centriole bound to the nuclear membrane

INTERACTIONS of natural and synthetic polyanions with chromatin are well documented, both at the structural and functional level. Frenster1 has shown that polyanions can enhance RNA transcription of chromatin, and this has been further studied by Chambon et al.2,3. Kraemer and Coffey4 have established that morphological modifications such as swelling of nuclei, loosening of the chromatin, and nuclear DNA release correlate with polyanion action5. All these phenomena have been logically interpreted as interactions of polyanions with histones leading to the release of genetic restrictions that histones produce, and Miller et al.6 and Berlowitz et al.7 have shown that selective binding of histones occurs. Arnold et al.8 have made an extensive morphological study of the structural alterations that are produced in nuclei by those polyanions which are capable of removing template restrictions. In all cases, molecular weights of the polyanions seem to be important4–8. As natural nuclear polyanions are known to occur, several theories have been proposed concerning the biological relevance of the above data1–9.

Action of Heparin on Nuclei: Solubilization of Chromatin enabling the Isolation of Nuclear Membranes

Sera of normal nonimmunized rabbits, pigs, calves, and humans contain tubulin-reactive antibodies Usually, low amounts of antibodies against tubulin of the IgG class (25-4 mg/100 ml of serum from nonimmunized animals) were isolated Anti-tubulin antibodies were also produced by injecting pig tubulin in complete Freund's adjuvant into rabbits Slightly higher amounts of anti-tubulin antibody were isolated from sera of immunized rabbits (7 mg/100 ml of serum) The cytoplasmic network of microtubules of Tcc 36 mouse cells in culture was not clearly stained by natural anti-tubulin antibodies, but dense staining of the centrosphere was observed In contrast, induced anti-tubulin antibodies densely stained cytoplasmic microtubular networks Vinblastine-induced tubulin paracrystals were equally stained by natural and induced anti-tubulin antibodies

Antibodies to tubulin in normal nonimmunized animals.

The mode of binding of 125I-labelled concanavalin A and succinyl-concanavalin A to rat thymocytes at 4 degrees C was investigated. Simultaneously, the free binding sites of the cell-bound lectin molecules were quantified by horseradish peroxidase binding. Concanavalin A showed cooperative binding while succinyl-concanavalin A did not. The number of molecules of concanavalin A bound to the cell surface when it was saturated was twice the number of molecules of succinyl-concanavalin A. We interpret these results as showing that the binding of native concanavalin A to thymocytes at 4 degrees C brings about a cooperative modification of the membrane which leads to appearance of new receptors. Divalent succinyl-concanavalin A has no such effect. Horseradish peroxidase binding to cell-bound lectin was shown to be related to the immobilization of membrane receptors; the more they are immobilized, the more receptor-associated lectin can bind horseradish peroxidase. This allowed us to establish that post-binding events, which we called micro-redistribution, occurred at 4 degrees C when either concanavalin A or succinyl-concanavalin A binds to cells. A cooperative restriction of the micromobility of cell receptors is produced by increasing concentrations of concanavalin A. Succinyl-concanavalin A does not restrict cell receptor mobility at any concentration tested. The results are discussed in terms of cell stimulation and cell agglutination.

https://febs.onlinelibrary.wiley.com/doi/pdf/10.1111/j.1432-1033.1976.tb10389.x

Cooperative Binding of Concanavalin A to Thymocytes at 4°C and Micro‐redistribution of Concanavalin A Receptors

An association between mitochondria and microtubules has been suggested from microscopic studies1,2 and by the cellular redistribution of mitochondria that is induced by colcemid, which disrupts microtubules1. Intermediate filaments have also been proposed to interact with mitochondria1. To investigate this association further, we have now studied the effect on the cytoskeleton of impairment of mitochondrial functions induced by three metabolic inhibitors: sodium azide, which inhibits electron transport; oligomycin, which inhibits ATP synthetase; and carbonylcyanide p-trifluoromethoxyphenylhydrazone (FCCP), a proton ionophore which uncouples electron transport from ATP synthesis. We have found that treatment of cells with FCCP leads to a disruption of microtubules and an aggregation of vimentin filaments. This effect is not provoked by mitochondrial ATP depletion, but could be linked to another event related to the rapid dissipation of the mitochondrial electrochemical gradient.

Michel Bornens

Papers

Is the centriole bound to the nuclear membrane

Action of Heparin on Nuclei: Solubilization of Chromatin enabling the Isolation of Nuclear Membranes

Antibodies to tubulin in normal nonimmunized animals.

Cooperative Binding of Concanavalin A to Thymocytes at 4°C and Micro‐redistribution of Concanavalin A Receptors

Reorganization of HeLa cell cytoskeleton induced by an uncoupler of oxidative phosphorylation.