Evaluación clínico-microbiológica de la asociación colistina/ rifampicina

Mitochondria were originally considered to be discrete, bean shaped organelles and are still commonly depicted in this way in scientific textbooks. However, it has now be known for some time that the mitochondria are in fact highly dynamic organelles, constantly undergoing fission and fusion (Bereiter-Hahn, 1978; Bereiter-Hahn and Voth, 1994). The balance of these determines their morphology and subsequently function. The frequency of these events alters mitochondrial morphology and in turn mitochondrial morphology can be used to predict the likelihood of fission or fusion events (Westrate et al., 2014). Maintaining the balance is crucial and may shift dependent on cellular requirements. With increased mitochondrial fusion increasing mtDNA stability (Chen et al., 2010) but allowing functional complementation to mitigate cellular stress and fission allowing selective removal of damaged

mitochondria (Youle and van der Bliek, 2012). Defects in fission and fusion have been associated with numerous human diseases e.g. Parkinson’s disease (Van Laar and Berman, 2009), Alzheimer’s disease (Santos et al., 2010) and Charcot-Marie-Tooth (CMT) disease (Palau et al., 2009).

Mitochondrial dynamics have been found to vary substantially between different cell types (Kuznetsov et al., 2009), with some cell types such as neurons having highly dynamic mitochondria while skeletal and cardiac mitochondria are less so. To date, the understanding of how frequently fission and fusion events occur in intact

myofibres is limited and cultured myoblasts and myotubes do not have the regular lattice-like structure of mitochondrial arrangement found in fully developed muscle so are unlikely to be representative of in vivo mitochondrial dynamics. So far, the best models of mitochondria dynamics in skeletal muscle are live cell imaging of intact mouse muscle (Pham et al., 2012), demonstrating that mitochondria are dynamic and fusion competent despite their static appearance.

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1.1.1 Fission

Fission is the process by which one mitochondrion divides to form two mitochondria (Figure 1.2). Early work to identify the fission machinery was completed in yeast where Dnm1 and Fis1 were identified (Bleazard et al., 1999; Mozdy et al., 2000).

Mammalian homologues of these proteins, dynamin related protein 1 (Drp1) and mitochondrial fission 1 protein (Fis1), were subsequently identified (Smirnova et al., 2001; James et al., 2003). Functional studies have demonstrated that knockdown of either protein causes mitochondria to elongate and become hyperfused whilst over expression fragments the mitochondrial network (Lee et al., 2004).

Figure 1.2 Mitochondrial fission. Schematic representing mitochondrial fission and demonstrating the localisation of Dynamin related protein 1(Drp1) and mitochondrial fission 1 protein (Fis1). Fis1 is found to be evenly distributed across the full OMM and may play a role in Drp1 recruitment. Drp1 forms a helix around the mitochondrion.

Contraction of this helix septates the membranes forming two mitochondria.

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In mammals, Drp1 is recruited from the cytosol and oligomerises forming a helix which, through the process of GTP hydrolysis constricts the mitochondrion ultimately resulting in membrane scission (Legesse-Miller et al., 2003; Mears et al., 2011). Drp1 recruitment and function are highly regulated by a number of post translational

modifications (Chang and Blackstone, 2010). Furthermore, numerous proteins have been found to interact with Drp1 and are thus thought to play a role in its recruitment.

These include Mff, Fis1, MID49 and MID51 (Mozdy et al., 2000; Loson et al., 2013).

Recently, Septin 2 was found to localise to fission sites and interact with Drp1, depletion of Septin 2 significantly decreases the recruitment of Drp1 thus confirming its importance (Pagliuso et al., 2016). The exact mechanism and roles that all of the proteins mentioned above play is not yet completely resolved.

Mitochondrial fission is important for the isolation and selective degradation of damaged mitochondria by mitophagy. A reduction in membrane potential results in fission, if the membrane potential remains low the mitochondrion cannot fuse back into the mitochondrial network and is targeted for mitophagy (Twig et al., 2008), as described in 1.4.2.

Due to the double membrane of the mitochondria, the fission process is a two-step process, requiring independent fission of the inner and outer membranes. Although the mechanism of mitochondrial fission is not yet completely understood, the proteins involved are well characterised.

1.1.2 Fusion

Fusion is the process by which two mitochondria combine to form a single

mitochondrion with continuous inner and outer membranes (Figure 1.3). OMM fusion is regulated by GTPases MFN1 and MFN2, which form homo- or heterodimers (Chen et al., 2003), that are all competent for fusion (Chen et al., 2005). Both MFN1 and MFN2 perform similar roles but are both required for successful fusion, though they have differential importance in different tissues (Chen et al., 2003; Chen et al., 2005).

MFN2 has also been demonstrated to perform a number of additional roles for which MFN1 cannot compensate e.g. tethering of mitochondria to the endoplasmic

reticulum (de Brito and Scorrano, 2008) and normal glucose homeostasis (Sebastian et al., 2012). The process of OMM fusion requires hydrolysis of GTP by the GTPase

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domain, which induces a conformational change allowing the membranes to fuse (Chan, 2006).

IMM fusion is thought to be mediated by OPA1 (Cipolat et al., 2004), which localises to the IMM. OPA1 undergoes alternative splicing and thus can exist in a number of functionally different isoforms (Delettre et al., 2001). Upon low membrane potential or low ATP levels, Oma1 is activated leading to the proteolytic cleavage of Opa1 and preventing fusion (Head et al., 2009).

Loss of the fusion machinery has been associated with disease in a number of tissues including heart (Papanicolaou et al., 2011), brain (Chen et al., 2007) and muscle (Chen et al., 2010). However, the importance of the fusion machinery varies in a tissue specific manner, as such some defects, such as knockout of MFN2 in kidney, only result in a phenotype when the mitochondria are stressed (Gall et al., 2012).

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Figure 1.3 Mitchondrial fusion. Schematic representing mitochondrial fusion and demonstrating the localisation of Opa1 and Mfn1/2. Binding of Mfn1/2 to the

membrane and dimerisation is necessary for tethering of the two mitochondria prior to GTP hydrolysis and fusion of the outer mitochondrial membrane. Opa1 mediates fusion of the inner mitochondrial membrane.HR1 and HR2; heptad repeat region 1 and 2.

1.1.3 Mitochondrial transport

Along with fission and fusion, mitochondrial dynamics also include small Brownian-like movements and transport over longer distances within the cell. However, the degree of motility varies on a tissue-specific basis, in some cells such as neurons and pancreatic cells, mitochondria can move quickly and continuously (Boldogh and Pon, 2007). Mitochondrial transport is particularly important in neurons, where it is used to balance changing energy needs of the subcellular locations (Hollenbeck and Saxton, 2005).

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In neurons anterograde mitochondrial transport takes mitochondria from the cell body into the axon towards the synapse and is generated by Kinesin-1(Hurd and Saxton, 1996; Glater et al., 2006), which interacts with Miro through adapter protein Milton (Stowers et al., 2002; Glater et al., 2006), in a calcium dependent manner (Wang and Schwarz, 2009). Retrograde transport of mitochondria back to the cell body is

achieved by a dynein motor (Martin et al., 1999).

However, in mature, cardiac and skeletal muscle, mitochondrial transport is not observed. In mice with photoactivatable mitochondria the mitochondrial appear immotile (Eisner et al., 2014) and in rat cardiomyocytes it has been demonstrated that mitochondria do not make large scale movements, only Brownian-like vibrations (Beraud et al., 2009). This may largely be due to the dependence of mitochondria on interactions with the cytoskeleton which gives the skeletal muscle mitochondria a crystal-like organisation (Vendelin et al., 2005). Specifically, mitochondria are known to be localised to the z-band in skeletal muscle and are tethered here by a protein complex containing desmin and plectin (described in more detail in 1.8.1), other members of this complex have yet to be identified.

Work in cultured myoblasts has demonstrated that many of the proteins required for transport of mitochondria in other cell types are also expressed in myoblasts,

although some kinesin heavy chain isoforms are not expressed (Iqbal and Hood, 2014). To date, this is the only work that has looked at mitochondrial transport in muscle and due to the change in cell architecture and mitochondrial positioning during myoblast differentiation and skeletal muscle development, the extent of mitochondrial transport in skeletal muscle in vivo is largely unknown.

1.4 Oxidative phosphorylation and ATP production 1.2.1 TCA cycle

The generation of ATP for cellular energy is a multi-step process starting with the anaerobic glycolysis, which feeds into the tricarboxylic acid cycle (TCA) or Kreb’s cycle in the mitochondrial matrix, before reaching the respiratory chain and oxidative phosphorylation. Anaerobic glycolysis Equation 1.1, which occurs in the cytosol, produces two molecules of pyruvate (Berg et al., 2012b). Pyruvate is either imported

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into mitochondria, to the Kreb’s cycle, or used to restore cytoplasmic NAD⁺ pools for further glycolysis. The process of creating NAD⁺ is either via fermentation in yeast or reduction of pyruvate to lactate by lactate dehydrogenase.

Glucose + 2[NAD⁺] + 2[ADP] + 2[Pi] → 2 [pyruvate] 2[NADH] + 2[H⁺] + 2[ATP] + 2[H²O]