Keratin-containing inclusions affect cell morphology and distribution of cytosolic cellular components

Many neurodegenerative diseases are characterized by the presence of protein aggregates bundled with intermediate filaments (IFs) and similar structures, known as Mallory bodies (MBs), are observed in various liver diseases. IFs are anchored at desmosomes and hemidesmosomes, however, interactions with other intercellular junctions have not been determined. We investigated the effect of IF inclusions on junction-associated and cytosolic proteins in various cultured cells. We performed gene transfection of the green fluorescent protein (GFP)-tagged cytokeratin (CK) 18 mutant arg89cys (GFP-CK18 R89C) in cultured cells and observed CK aggregations as well as loss of IF networks. Among various junction-associated proteins, zonula occludens-1 and beta-catenin were colocalized with CK aggregates on immunofluorescent analyses. Similar results were obtained on immunostaining for cytosolic proteins, 14-3-3 zeta protein, glucose-6-phosphate dehydrogenase and DsRed. E-cadherin, a basolateral membrane protein in polarized epithelia, was present on both the apical and basolateral domains in GFP-CK18 R89C-transfected cells. Furthermore, cells containing CK aggregates were significantly larger than GFP-tagged wild type CK18 (GFP-WT CK18)-transfected or non-transfected cells (P < 0.01) and sometimes their morphology was significantly altered. Our data indicate that CK aggregates affect not only cell morphology but also the localization of various cytosolic components, which may affect the cellular function.     

MIRO1 influences the morphology and intracellular distribution of mitochondria during embryonic cell division in Arabidopsis

Regulating the morphology and intracellular distribution of mitochondria is essential for embryo development in animals. However, the importance of such regulation is not clearly defined in plants. The evolutionarily conserved Miro proteins are known to be involved in the regulation of mitochondrial morphology and motility. We previously demonstrated that MIRO1, an Arabidopsis thaliana orthologue of the Miro protein, is required for embryogenesis. An insertional mutation in the MIRO1 gene causes arrest of embryonic cell division, leading to abortion of the embryo at an early stage. Here we investigated the role of MIRO1 in the regulation of mitochondrial behaviour in egg cells and early-stage embryos using GFP-labeled mitochondria. Two-photon laser scanning microscopy revealed that, in miro1 mutant egg cells, mitochondria are abnormally enlarged, although egg cell formation is nearly unaffected. After fertilization and subsequent zygotic cell division, the homozygous miro1 mutant two-celled embryo contained a significantly reduced number of mitochondria in its apical cell compared with the wild type, suggesting that the miro1 mutation inhibits proper intracellular distribution of mitochondria, leading to an arrest of embryonic cell division. Our findings suggest that proper mitochondrial morphology and intracellular distribution are maintained by MIRO1 and are vital for embryonic cell division.     

Parkinson's disease-associated DJ-1 mutations impair mitochondrial dynamics and cause mitochondrial dysfunction

Mitochondrial dysfunction represents a critical event during the pathogenesis of Parkinson's disease (PD) and expanding evidences demonstrate that an altered balance in mitochondrial fission/fusion is likely an important mechanism leading to mitochondrial and neuronal dysfunction/degeneration. In this study, we investigated whether DJ-1 is involved in the regulation of mitochondrial dynamics and function in neuronal cells. Confocal and electron microscopic analysis demonstrated that M17 human neuroblastoma cells over-expressing wild-type DJ-1 (WT DJ-1 cells) displayed elongated mitochondria while M17 cells over-expressing PD-associated DJ-1 mutants (R98Q, D149A and L166P) (mutant DJ-1 cells) showed significant increase of fragmented mitochondria. Similar mitochondrial fragmentation was also noted in primary hippocampal neurons over-expressing PD-associated mutant forms of DJ-1. Functional analysis revealed that over-expression of PD-associated DJ-1 mutants resulted in mitochondria dysfunction and increased neuronal vulnerability to oxidative stress (H(2) O(2)) or neurotoxin. Further immunoblot studies demonstrated that levels of dynamin-like protein (DLP1), also known as Drp1, a regulator of mitochondrial fission, was significantly decreased in WT DJ-1 cells but increased in mutant DJ-1 cells. Importantly, DLP1 knockdown in these mutant DJ-1 cells rescued the abnormal mitochondria morphology and all associated mitochondria/neuronal dysfunction. Taken together, these studies suggest that DJ-1 is involved in the regulation of mitochondrial dynamics through modulation of DLP1 expression and PD-associated DJ-1 mutations may cause PD by impairing mitochondrial dynamics and function.     

Vimentin intermediate filaments protect mitochondria from oxidative stress

The role of intermediate filaments (IFs) in eukaryotic cells is still unclear. The disturbance of mitochondria distribution and function, in particular the enhanced production of reactive oxygen species (ROS) and decreased membrane potential, is observed in cells devoid of IFs. The aim of this work was to study the dependence of mitochondria sensitivity to oxidative stress on the presence of vimentin IFs. It was found that mitochondria are less sensitive to ROS in cells containing vimentin than in cells devoid of vimentin. Besides, mitochondrial membrane potential was demonstrated to increase upon regeneration of vimentin IFs in the cells. Substitution of Pro-57 by Arg in N-terminal part of the vimentin molecule abandoned its protective ability and the effect on mitochondrial membrane potential.     

Altered fusion dynamics underlie unique morphological changes in mitochondria during hypoxia-reoxygenation stress

Functional states of mitochondria are often reflected in characteristic mitochondrial morphology. One of the most fundamental stress conditions, hypoxia-reoxygenation has been known to cause impaired mitochondrial function accompanied by structural abnormalities, but the underlying mechanisms need further investigation. Here, we monitored bioenergetics and mitochondrial fusion-fission in real time to determine how changes in mitochondrial dynamics contribute to structural abnormalities during hypoxia-reoxygenation. Hypoxia-reoxygenation resulted in the appearance of shorter mitochondria and a decrease in fusion activity. This fusion inhibition was a result of impaired ATP synthesis rather than Opa1 cleavage. A striking feature that appeared during hypoxia in glucose-free and during reoxygenation in glucose-containing medium was the formation of donut-shaped (toroidal) mitochondria. Donut formation was triggered by opening of the permeability transition pore or K(+) channels, which in turn caused mitochondrial swelling and partial detachment from the cytoskeleton. This then favored anomalous fusion events (autofusion and fusion at several sites among 2-3 mitochondria) to produce the characteristic donuts. Donuts effectively tolerate matrix volume increases and give rise to offspring that can regain ΔΨ(m). Thus, the metabolic stress during hypoxia-reoxygenation alters mitochondrial morphology by inducing distinct patterns of mitochondrial dynamics, which includes processes that could aid mitochondrial adaptation and functional recovery.     

The Dynamin-related GTPase,Dnm1p,Controls Mitochondrial Morphology in Yeast

The Saccharomyces cerevisiae Dnm1 protein is structurally related to dynamin, a GTPase required for membrane scission during endocytosis. Here we show that Dnm1p is essential for the maintenance of mitochondrial morphology. Disruption of the DNM1 gene causes the wild-type network of tubular mitochondrial membranes to collapse to one side of the cell but does not affect the morphology or distribution of other cytoplasmic organelles. Dnm1 proteins containing point mutations in the predicted GTP-binding domain or completely lacking the GTP-binding domain fail to rescue mitochondrial morphology defects in a dnm1 mutant and induce dominant mitochondrial morphology defects in wild-type cells. Indirect immunofluorescence reveals that Dnm1p is distributed in punctate structures at the cell cortex that colocalize with the mitochondrial compartment. These Dnm1p-containing structures remain associated with the spherical mitochondria found in an mdm10 mutant strain. In addition, a portion of Dnm1p cofractionates with mitochondrial membranes during differential sedimentation and sucrose gradient fractionation of wild-type cells. Our results demonstrate that Dnm1p is required for the cortical distribution of the mitochondrial network in yeast, a novel function for a dynamin-related protein.     

Mitochondria provide the main source of cytosolic ATP for activation of outward-rectifying K+ channels in mesophyll protoplast of chlorophyll-deficient mutant rice (OsCHLH) seedlings

The role of mitochondria in providing intracellular ATP that controls the activity of plasma membrane outward-rectifying K+ channels was evaluated. The OsCHLH rice mutant, which lacks chlorophyll in the thylakoids, was isolated by T-DNA gene trapping (Jung, K.-H., Hur, J., Ryu, C.-H., Choi, Y., Chung, Y.-Y., Miyao, A., Hirochika, H., and An, G. (2003) Plant Cell Physiol. 44, 463-472). The OsCHLH mutant is unable to fix CO2 and exhibits reduced growth. Wild type and mutant plants exhibit similar rates of respiratory O2 uptake in the dark, whereas the rate of photosynthetic O2 evolution by the mutant was negligible during illumination. During dark respiration the wild type and mutant exhibited similar levels of cytoplasmic ATP. In the mutant oligomycin treatment (an inhibitor of mitochondrial F1F0-ATPase) drastically reduced ATP production. The fact that this was reversed by the addition of glucose suggested that the mutant produced ATP exclusively from mitochondria but not from chloroplasts. In whole cell patch clamp experiments, the activity of outward-rectifying K+ channels of rice mesophyll cells showed ATP-dependent currents, which were 1.5-fold greater in wild type than in mutant cells. Channels in both wild type and mutant cells were deactivated by the removal of cytosolic ATP, whereas in the presence of ATP the channels remained active. We conclude that mesophyll cells in the OsCHLH rice mutant derive ATP from mitochondrial respiration, and that this is critical for the normal function of plasma membrane outward-rectifying K+ channels.     

Effects of antimitotic and antimitochondrial agents on the cellular distribution of microtubules and mitochondria

Using antibodies to a mitochondrial molecular chaperone class of protein, which is specifically altered in mutants resistant to microtubule (MT) inhibitors, the effect of a number of MT and mitochondrial inhibitors on the cellular distribution of mitochondria and various cytoskeletal filaments was examined. Treatment of Chinese hamster ovary (CHO) or chicken embryo fibroblast (CEF) cells with the MT inhibitors podophyllotoxin, colchicine, nocodazole and vinblastine caused depolymerization of cellular MTs, but had no significant effect on the distribution patterns of mitochondria. This is attributed to the association of mitochondria with intermediate filaments (IFs) which are not destroyed under these conditions. In contrast to MT inhibitors, treatment of CEFs with the potassium ionophores nonactin and valinomycin caused aggregation of mitochondria towards the perinuclear region of the cells, without having any apparent effect on cellular MTs. This observation suggests that mitochondrial membrane potential, which is abolished by these drugs, play a role in the cellular distribution of mitochondria. In cells recovering from the effects of MT inhibitors, mitochondria have been found to surround the MT organizing complexes and upon complete recovery a realignment of MTs with mitochondria takes place. These observations suggest that MT growth in cells does not occur in a completely random manner but that mitochondria may play some role in their directional growth.     

Muscle intermediate filaments and their links to membranes and membranous organelles

Intermediate filaments (IFs) play a key role in the integration of structure and function of striated muscle, primarily by mediating mechanochemical links between the contractile apparatus and mitochondria, myonuclei, the sarcolemma and potentially the vesicle trafficking apparatus. Linkage of all these membranous structures to the contractile apparatus, mainly through the Z-disks, supports the integration and coordination of growth and energy demands of the working myocyte, not only with force transmission, but also with de novo gene expression, energy production and efficient protein and lipid trafficking and targeting. Desmin, the most abundant and intensively studied muscle intermediate filament protein, is linked to proper costamere organization, myoblast and stem cell fusion and differentiation, nuclear shape and positioning, as well as mitochondrial shape, structure, positioning and function. Similar links have been established for lysosomes and lysosome-related organelles, consistent with the presence of widespread links between IFs and membranous structures and the regulation of their fusion, morphology and stabilization necessary for cell survival.     

Loss of Yme1L perturbates mitochondrial dynamics

Yme1L is an AAA protease that is embedded in the mitochondrial inner membrane with its catalytic domain facing the mitochondrial inner-membrane space. However, how Yme1L regulates mammalian mitochondrial function is still obscure. We find that endogenous Yme1L locates at punctate structures of mitochondria, and that loss of Yme1L in mouse embryonic fibroblast (MEF) cells results in mitochondrial fragmentation and leads to significant increased ‘kiss-and-run'' type of mitochondrial fusion; however, Yme1L knockdown (shYme1L (short hairpin-mediated RNA interference of Yme1L)) cells still remain normal mitochondrial fusion although shYme1L mitochondria have a little bit less fusion and fission rates, and the shYme1L-induced fragmentation is due to a little bit more mitochondrial fission than fusion in cells. Furthermore, shYme1L-induced mitochondrial fragmentation is independent on optic atrophy 1 (OPA1) S1 or S2 processing, and shYme1L results in the stabilization of OPA1 long form (L-OPA1); in addition, the exogenous expression of OPA1 or L-OPA1 facilitates the shYme1L-induced mitochondrial fragmentation, thus this fragmentation induced by shYme1L appears to be associated with L-OPA1''s stability. ShYme1L also causes a slight increase of mitochondrial dynamics proteins of 49 kDa and mitochondrial fission factor (Mff), which recruit mitochondrial key fission factor dynamin-related protein 1 (Drp1) into mitochondria in MEF cells, and loss of Drp1 or Mff inhibits the shYme1L-induced mitochondrial fragmentation. In addition, there is interaction between SLP-2 with Yme1L and shYme1L cells retain stress-induced mitochondrial hyperfusion. Taken together, our results clarify how Yme1L regulates mitochondrial morphology.     

Switch in Fas-activated death signaling pathway as result of keratin 8/18-intermediate filament loss

Fas-induced apoptosis is initiated through the recruitment of FADD and procaspase 8 to form the death-inducing signaling complex (DISC). In some cells (type I cells) the initiator caspase 8 directly activates effector caspases such as procaspase 3, whereas in others (type II cells) the death signal is amplified through mitochondria. In epithelial cells, Fas-induced hierarchic caspase activation is also linked with DEDD, a member of the DED family that binds to keratin (K) intermediate filaments (IFs). Hepatocytes are type II cells and their IFs are made exclusively of K8/K18. We have shown previously that K8-null mouse hepatocytes, lacking K8/K18 IFs, are more sensitive than their wild-type counterparts to Fas-induced apoptosis. Here, by examining the cell-death kinetics and death-signaling ordering, we found that K8-null hepatocytes exhibited prominent DISC formation, higher procaspase 8 activation and direct procaspase 3 activation as reported for type I cells; however they experienced a reduced Bid cleavage and a stronger procaspase 9 activation. In addition, the K8/K18 loss altered the DEDD ubiquitination status and nuclear/cytoplasmic distribution. Together, the results suggest that the K8/K18 loss induces a switch in Fas-induced death signaling, likely through a DEDD involvement. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Keratin-protein kinase C interaction in reactive oxygen species-induced hepatic cell death through mitochondrial signaling

Keratins (Ks), the intermediate filament (IF) proteins of epithelia, constitute at least 20 cytoskeletal proteins subdivided into type I (K9-20) and type II (K1-K8) and expressed as type I/type II pairs in a cell differentiation manner. Hepatocyte IFs are made only of K8/K18, the hallmark of simple epithelial cells. We have shown previously that a K8/K18 loss leads to a modulation of apoptosis in Fas-stimulated mouse hepatocytes. Here we report that K8-knockout mouse hepatocytes and K8-knockdown H4-II-E-C3 (shK8b1) rat hepatoma cells were much more resistant than their K8/K18-containing counterparts, wild-type hepatocytes, and H4ev hepatoma cells, in response to excess H2O2 or tert-butyl hydroperoxide, a ROS generator. While excess H2O2 altered glutathione (GSH) and ROS levels in H4ev versus shK8b1 cells, the differential death response was largely GSH level independent. Assessment of key cell death features revealed that hepatic cells exposed to H2O2 die through a mitochondrial involvement. Similarly, administration of the GSH depletor L-buthionine-sulfoximine to generate mitochondrial ROS-sensitized H4-II-E-C3 cells but not shK8b1 cells to death. Treatment with protein kinase C (PKC) inhibitors yielded a resistance of H2O2-treated H4-II-E-C3 cells comparable to that of nontreated shK8b1 cells, which in turn were not affected by the treatment. In addition, this differential death response was associated with altered PKCdelta activation and surface-membrane/mitochondria distribution in H2O2-treated shK8b1 cells. Together, these results point to a key regulatory function for K8/K18 in ROS-induced mitochondria-mediated death through PKCdelta involvement in hepatic cells.     

Treatment of human cells derived from MERRF syndrome by peptide-mediated mitochondrial delivery

Background aimsThe feasibility of delivering mitochondria using the cell-penetrating peptide Pep-1 for the treatment of MERRF (myoclonic epilepsy with ragged red fibers) syndrome, which is caused by point mutations in the transfer RNA genes of mitochondrial DNA, is examined further using cellular models derived from patients with MERRF syndrome.MethodsHomogenesis of mitochondria (wild-type mitochondria) isolated from normal donor cells with about 83.5% preserved activity were delivered into MERRF fibroblasts by Pep-1 conjugation (Pep-1-Mito).ResultsDelivered doses of 52.5 μg and 105 μg Pep-1-Mito had better delivered efficiency and mitochondrial biogenesis after 15 days of treatment. The recovery of mitochondrial function in deficient cells receiving 3 days of treatment with peptide-mediated mitochondrial delivery was comprehensively demonstrated by restoration of oxidative phosphorylation subunits (complex I, III and IV), mitochondrial membrane potential, adenosine triphosphate synthesis and reduction of reactive oxygen species production. The benefits of enhanced mitochondrial regulation depended on the function of foreign mitochondria and not the existence of mitochondrial DNA and can be maintained for at least 21 days with dramatically elongated mitochondrial morphology. In contrast to delivery of wild-type mitochondria, the specific regulation of Pep-1-Mito during MERRF syndrome progression in cells treated with mutant mitochondria was reflected by the opposite performance, with increase in reactive oxygen species production and matrix metalloproteinase activity.ConclusionsThe present study further illustrates the feasibility of mitochondrial intervention therapy using the novel approach of peptide-mediated mitochondrial delivery and the benefit resulting from mitochondria-organelle manipulation.     

Modulation of mitochondrial function and morphology by interaction of Omi/HtrA2 with the mitochondrial fusion factor OPA1

Loss of Omi/HtrA2 function leads to nerve cell loss in mouse models and has been linked to neurodegeneration in Parkinson's and Huntington's disease. Omi/HtrA2 is a serine protease released as a pro-apoptotic factor from the mitochondrial intermembrane space into the cytosol. Under physiological conditions, Omi/HtrA2 is thought to be involved in protection against cellular stress, but the cytological and molecular mechanisms are not clear. Omi/HtrA2 deficiency caused an accumulation of reactive oxygen species and reduced mitochondrial membrane potential. In Omi/HtrA2 knockout mouse embryonic fibroblasts, as well as in Omi/HtrA2 silenced human HeLa cells and Drosophila S2R+ cells, we found elongated mitochondria by live cell imaging. Electron microscopy confirmed the mitochondrial morphology alterations and showed abnormal cristae structure. Examining the levels of proteins involved in mitochondrial fusion, we found a selective up-regulation of more soluble OPA1 protein. Complementation of knockout cells with wild-type Omi/HtrA2 but not with the protease mutant [S306A]Omi/HtrA2 reversed the mitochondrial elongation phenotype and OPA1 alterations. Finally, co-immunoprecipitation showed direct interaction of Omi/HtrA2 with endogenous OPA1. Thus, we show for the first time a direct effect of loss of Omi/HtrA2 on mitochondrial morphology and demonstrate a novel role of this mitochondrial serine protease in the modulation of OPA1. Our results underscore a critical role of impaired mitochondrial dynamics in neurodegenerative disorders.     

Phospholipid scramblase 3 controls mitochondrial structure, function, and apoptotic response

Phospholipid scramblase 3 (PLS3) is a newly recognized member of a family of proteins responsible for phospholipid translocation between two lipid compartments. To study PLS3 function in mitochondria, we disrupted its conserved calcium-binding motif yielding an inactive mutant PLS3(F258V). Cells transfected with PLS3(F258V) exhibited reduced proliferative capacity. Mitochondrial analysis revealed that PLS3(F258V)-expressing cells have decreased mitochondrial mass shown by lower cytochrome c and cardiolipin (CL) content, poor mitochondrial respiration, and reduced oxygen consumption and intracellular ATP; whereas wild-type PLS3-transfected cells exhibit increased mitochondrial mass and enhanced respiration. Electron microscopic examination revealed that the mitochondria in PLS3(F258V)-expressing cells have densely packed cristae and are fewer in number and larger than those in control cells. The abnormal mitochondrial metabolism and structure in PLS3(F258V)-expressing cells were associated with decreased sensitivity to UV- and tBid-induced apoptosis and diminished translocation of CL to the mitochondrial outer membrane. In contrast, wild-type PLS3-transfected cells displayed increased sensitivity to apoptosis and enhanced CL translocation. These studies identify PLS3 as a critical regulator of mitochondrial structure and respiration, and CL transport in apoptosis.     

Ezrin functionality and hypothermic preservation injury in LLC-PK1 cells

Renal epithelial cells from donor kidneys are susceptible to hypothermic preservation injury, which is attenuated when they over express the cytoskeletal linker protein ezrin. This study was designed to characterize the mechanisms of this protection. Renal epithelial cell lines were created from LLC-PK1 cells, which expressed mutant forms of ezrin with site directed alterations in membrane binding functionality. The study used cells expressing wild type ezrin, T567A, and T567D ezrin point mutants. The A and D mutants have constitutively inactive and active membrane binding conformations, respectively. Cells were cold stored (4 °C) for 6-24 h and reperfused for 1h to simulate transplant preservation injury. Preservation injury was assessed by mitochondrial activity (WST-1) and LDH release. Cells expressing the active ezrin mutant (T567D) showed significantly less preservation injury compared to wild type or the inactive mutant (T567A), while ezrin-specific siRNA knockdown and the inactive mutant potentiated preservation injury. Ezrin was extracted and identified from purified mitochondria. Furthermore, isolated mitochondria specifically bound anti-ezrin antibodies, which were reversed with the addition of exogenous recombinant ezrin. Recombinant wild type ezrin significantly reduced the sensitivity of the mitochondrial permeability transition pore (mPTP) to calcium, suggesting ezrin may modify mitochondrial function. In conclusion, the cytoskeletal linker protein ezrin plays a significant role in hypothermic preservation injury in renal epithelia. The mechanisms appear dependent on the molecule's open configuration (traditional linker functionality) and possibly a novel mitochondrial specific role, which may include modulation of mPTP function or calcium sensitivity.     

Inactivation of the mitochondrial heat shock protein zim17 leads to aggregation of matrix hsp70s followed by pleiotropic effects on morphology and protein biogenesis

The biogenesis of mitochondrial matrix proteins involves the translocase of the outer membrane, the presequence translocase of the inner membrane and the presequence translocase-associated motor. The mitochondrial heat shock protein 70 (mtHsp70) forms the central core of the motor. Recent studies led to the identification of Zim17, a mitochondrial zinc finger motif protein that interacts with mtHsp70. Different views have been reported on the localization of Zim17 in the mitochondrial inner membrane or matrix. Depletion of Zim17 impairs several critical mitochondrial processes, leading to inhibition of protein import, defects of Fe/S protein biogenesis and aggregation of Hsp70s in the matrix. Additionally, we found that inactivation of Zim17 altered the morphology of mitochondria. These pleiotropic effects raise the question of the specific function of Zim17 in mitochondria. Here, we report that Zim17 is a heat shock protein of the mitochondrial matrix that is loosely associated with the inner membrane. To address the function of Zim17 in organello, we generated a temperature-sensitive mutant allele of the ZIM17 gene in yeast. Upon a short-term shift of the yeast mutant cells to a non-permissive temperature, matrix Hsp70s aggregated while protein import, Fe/S protein activity and mitochondrial morphology were not, or only mildly, affected. Only after a long-term shift to non-permissive temperature, were strong defects in protein import, Fe/S protein activity and mitochondrial morphology observed. These findings suggest that the heat shock protein Zim17 plays a specific role in preventing protein aggregation in the mitochondrial matrix, and that aggregation of Hsp70s causes pleiotropic effects on protein biogenesis and mitochondrial morphology.     

Regulation of mitochondrial morphology and inheritance by Mdm10p,a protein of the mitochondrial outer membrane

Yeast cells with the mdm10 mutation possess giant spherical mitochondria and are defective for mitochondrial inheritance. The giant mitochondria display classical features of mitochondrial ultrastructure, yet they appear incapable of movement or division. Genetic analysis indicated that the mutant phenotypes resulted from a single nuclear mutation, and the isolated MDM10 gene restored wild-type mitochondrial distribution and morphology when introduced into mutant cells. MDM10 encodes a protein of 56.2 kD located in the mitochondrial outer membrane. Depletion of Mdm10p from cells led to a condensation of normally extended, tubular mitochondria into giant spheres, and reexpression of the protein resulted in a rapid restoration of normal mitochondrial morphology. These results demonstrate that Mdm10p can control mitochondrial morphology, and that it plays a role in the inheritance of mitochondria.     

Role of H(abc) domain in membrane trafficking and targeting of syntaxin 1A

Membrane syntaxin plays essential roles in exocytosis in eukaryotic cells. The conservative H(abc) domain in plasma membrane syntaxins implies important roles for syntaxin targeting and function. Our previous study showed H(abc) domain was necessary for the trafficking and cluster distribution of syntaxin 1A on the plasma membrane. Here we identified which of the three domains (H(a), H(b) and H(c)) was essential for Stx1A trafficking and clustering. We found that, in INS-1 cells, the mutant truncated with either H(a), H(b) or H(c) domain could be sorted to the cell surface by a different mechanism compared to that of whole H(abc) truncated mutant. In contrast to wild type Stx1A, none of the mutants showed cluster distribution at the functional sites, suggesting that the physiological localization of Stx1A relies on intact H(abc) domain. Furthermore Munc18-1 is found not to be essential for Stx1A cluster distribution, despite important role in stabilizing membrane delivery of Stx1A.     

The basal flux of Akt in the mitochondria is mediated by heat shock protein 90

Akt is a known client protein of heat shock protein 90 (HSP90). We have found that HSP90 is responsible for Akt accumulation in the mitochondria in unstimulated cells. Treatment of SH-SY5Y neuroblastoma cells and human embryonic kidney cells with the HSP90 inhibitors novobiocin and geldanamycin caused substantial decreases in the level of Akt in the mitochondria without affecting the level of Akt in the cytosol. Moreover, intracerebroventricular injection of novobiocin into mice brains decreased Akt levels in cortical mitochondria. Knockdown of HSP90 expression with short interfering RNA also caused a significant decrease in Akt levels in the mitochondria without affecting total Akt levels. Using a mitochondrial import assay it was found that Akt is transported into the mitochondria. Furthermore, it was found that the mitochondrial import of Akt was independent of Akt activation as both an unmodified Akt and constitutively active mutant Akt; both readily accumulated in the mitochondria in an HSP90-dependent manner. Interestingly, incubation of isolated mitochondria with constitutively active Akt caused visible alterations in mitochondrial morphology, including pronounced remodeling of the mitochondrial matrix. This effect was blocked when Akt was mostly excluded from the mitochondria with novobiocin treatment. These results indicate that the level of Akt in the mitochondria is dependent on HSP90 chaperoning activity and that Akt import can cause dynamic changes in mitochondrial configuration.