Kasturi Mitra

Mitochondria Supply Membranes for Autophagosome Biogenesis during Starvation

Starvation-induced autophagosomes engulf cytosol and/or organelles and deliver them to lysosomes for degradation, thereby resupplying depleted nutrients. Despite advances in understanding the molecular basis of this process, the membrane origin of autophagosomes remains unclear. Here, we demonstrate that, in starved cells, the outer membrane of mitochondria participates in autophagosome biogenesis. The early autophagosomal marker, Atg5, transiently localizes to punctae on mitochondria, followed by the late autophagosomal marker, LC3. The tail-anchor of an outer mitochondrial membrane protein also labels autophagosomes and is sufficient to deliver another outer mitochondrial membrane protein, Fis1, to autophagosomes. The fluorescent lipid NBD-PS (converted to NBD-phosphotidylethanolamine in mitochondria) transfers from mitochondria to autophagosomes. Photobleaching reveals membranes of mitochondria and autophagosomes are transiently shared. Disruption of mitochondria/ER connections by mitofusin2 depletion dramatically impairs starvation-induced autophagy. Mitochondria thus play a central role in starvation-induced autophagy, contributing membrane to autophagosomes.

A hyperfused mitochondrial state achieved at G1–S regulates cyclin E buildup and entry into S phase

Mitochondria undergo fission–fusion events that render these organelles highly dynamic in cells. We report a relationship between mitochondrial form and cell cycle control at the G1–S boundary. Mitochondria convert from isolated, fragmented elements into a hyperfused, giant network at G1–S transition. The network is electrically continuous and has greater ATP output than mitochondria at any other cell cycle stage. Depolarizing mitochondria at early G1 to prevent these changes causes cell cycle progression into S phase to be blocked. Inducing mitochondrial hyperfusion by acute inhibition of dynamin-related protein-1 (DRP1) causes quiescent cells maintained without growth factors to begin replicating their DNA and coincides with buildup of cyclin E, the cyclin responsible for G1-to-S phase progression. Prolonged or untimely formation of hyperfused mitochondria, through chronic inhibition of DRP1, causes defects in mitotic chromosome alignment and S-phase entry characteristic of cyclin E overexpression. These findings suggest a hyperfused mitochondrial system with specialized properties at G1–S is linked to cyclin E buildup for regulation of G1-to-S progression.

Analysis of Mitochondrial Dynamics and Functions Using Imaging Approaches

Mitochondria are organelles that have been primarily known as the powerhouse of the cell. However, recent advances in the field have revealed that mitochondria are also involved in many other cellular activities like lipid modifications, redox balance, calcium balance, and even controlled cell death. These multifunctional organelles are motile and highly dynamic in shapes and forms; the dynamism is brought about by the mitochondrias ability to undergo fission and fusion with each other. Therefore, it is very important to be able to image mitochondrial shape changes to relate to the variety of cellular functions these organelles have to accomplish. The protocols described here will enable researchers to perform steady‐state and time‐lapse imaging of mitochondria in live cells by using confocal microscopy. High‐resolution three‐dimensional imaging of mitochondria will not only be helpful in understanding mitochondrial structure in detail but it also could be used to analyze their structural relationships with other organelles in the cell. FRAP (fluorescence recovery after photobleaching) studies can be performed to understand mitochondrial dynamics or dynamics of any mitochondrial molecule within the organelle. The microirradiation assay can be performed to study functional continuity between mitochondria. A protocol for measuring mitochondrial potential has also been included in this unit. In conclusion, the protocols described here will aid the understanding of mitochondrial structure‐function relationship. Curr. Protoc. Cell Biol. 46:4.25.1‐4.25.21.

Mitochondrial fission‐fusion as an emerging key regulator of cell proliferation and differentiation

Mitochondrial shape change, brought about by molecules that promote either fission or fusion between individual mitochondria, has been documented in several model systems. However, the deeper significance of mitochondrial shape change has only recently begun to emerge: among others, it appears to play a role in the regulation of cell proliferation. Here, I review the emerging interplay between mitochondrial fission‐fusion components with cell cycle regulatory machineries and how that may impact cell differentiation. Regulation of mitochondrial shape may modulate mitochondrial metabolism and/or energetics to promote crosstalk between signaling components and the cell cycle machinery. Focused research in this area will reveal the exact role of mitochondria in development and disease, specifically in stem cell regulation and tumorigenesis. Such research may also reveal whether and how the endosymbiotic event that gave rise to the mitochondrion was crucial for the evolution of cell cycle regulatory mechanisms in eukaryotes that are absent in prokaryotes.

Novel Tyrosine-Phosphorylated Post-Pyruvate Metabolic Enzyme, Dihydrolipoamide Dehydrogenase, Involved in Capacitation of Hamster Spermatozoa

Abstract Capacitation is a process that confers fertilizing ability to spermatozoa and this critical event occurs in the development of mammalian spermatozoa during their transit through the female reproductive tract and precedes fertilization. Because spermatozoa are relatively silent in transcription and translation, posttranslational modifications perform the regulatory functions in these cells during capacitation. In this report, we identify a candidate protein, dihydrolipoamide dehydrogenase, which is a post-pyruvate metabolic enzyme, exhibiting tyrosine phosphorylation during hamster spermatozoal capacitation. This is the first report showing dihydrolipoamide dehydrogenase as a phosphoprotein. The cDNA sequence of hamster testes dihydrolipoamide dehydrogenase does not show any variation from the already reported mammalian dihydrolipoamide dehydrogenases. Downregulation of the activity of the hamster spermatozoal enzyme by its specific inhibitor, 5-methoxyindole-2-carboxylic acid, blocks acrosome reaction completely and hyperactivation partially, confirming the role of dihydrolipoamide dehydrogenase in hamster spermatozoal capacitation. We also delineate the temporal involvement of glucose and pyruvate-lactate, showing that the former is required in the earlier stages and the latter for the later stages of hamster spermatozoal capacitation. The essentiality of pyruvate-lactate during hyperactivation and acrosome reaction necessitates the involvement of the post-pyruvate-lactate enzyme, dihydrolipoamide dehydrogenase.

DRP1-dependent mitochondrial fission initiates follicle cell differentiation during Drosophila oogenesis

Reduced Drp1-mediated mitochondrial fission decreases cell cycle exit and prevents Notch-dependent follicle cell differentiation during oogenesis.

Dysfunctional mitochondrial bioenergetics and oxidative stress in Akita(+/Ins2)-derived β-cells.

Insulin release from pancreatic β-cells plays a critical role in blood glucose homeostasis, and β-cell dysfunction leads to the development of diabetes mellitus. In cases of monogenic type 1 diabetes mellitus (T1DM) that involve mutations in the insulin gene, we hypothesized that misfolding of insulin could result in endoplasmic reticulum (ER) stress, oxidant production, and mitochondrial damage. To address this, we used the Akita(+/Ins2) T1DM model in which misfolding of the insulin 2 gene leads to ER stress-mediated β-cell death and thapsigargin to induce ER stress in two different β-cell lines and in intact mouse islets. Using transformed pancreatic β-cell lines generated from wild-type Ins2(+/+) (WT) and Akita(+/Ins2) mice, we evaluated cellular bioenergetics, oxidative stress, mitochondrial protein levels, and autophagic flux to determine whether changes in these processes contribute to β-cell dysfunction. In addition, we induced ER stress pharmacologically using thapsigargin in WT β-cells, INS-1 cells, and intact mouse islets to examine the effects of ER stress on mitochondrial function. Our data reveal that Akita(+/Ins2)-derived β-cells have increased mitochondrial dysfunction, oxidant production, mtDNA damage, and alterations in mitochondrial protein levels that are not corrected by autophagy. Together, these findings suggest that deterioration in mitochondrial function due to an oxidative environment and ER stress contributes to β-cell dysfunction and could contribute to T1DM in which mutations in insulin occur.

Metabolic Reprogramming Is Required for Myofibroblast Contractility and Differentiation

Background: Myofibroblasts, by virtue of their functions, are highly energy-dependent. Results: TGF-β1-induced myofibroblast differentiation is associated with a metabolic reprogramming. This metabolic adaptation is essential to the expression of myofibroblast-related genes. Conclusion: Metabolic reprogramming is a hallmark of myofibroblast differentiation and is critical for its contractile function. Significance: This is the first report that links bioenergetics to myofibroblast activation. Contraction is crucial in maintaining the differentiated phenotype of myofibroblasts. Contraction is an energy-dependent mechanism that relies on the production of ATP by mitochondria and/or glycolysis. Although the role of mitochondrial biogenesis in the adaptive responses of skeletal muscle to exercise is well appreciated, mechanisms governing energetic adaptation of myofibroblasts are not well understood. Our study demonstrates induction of mitochondrial biogenesis and aerobic glycolysis in response to the differentiation-inducing factor transforming growth factor β1 (TGF-β1). This metabolic reprogramming is linked to the activation of the p38 mitogen-activated protein kinase (MAPK) pathway. Inhibition of p38 MAPK decreased accumulation of active peroxisome proliferator-activated receptor γ coactivator 1α in the nucleus and altered the translocation of mitochondrial transcription factor A to the mitochondria. Genetic or pharmacologic approaches that block mitochondrial biogenesis or glycolysis resulted in decreased contraction and reduced expression of TGF-β1-induced α-smooth muscle actin and collagen α-2(I) but not of fibronectin or collagen α-1(I). These data indicate a critical role for TGF-β1-induced metabolic reprogramming in regulating myofibroblast-specific contractile signaling and support the concept of integrating bioenergetics with cellular differentiation.

Coordinated elevation of mitochondrial oxidative phosphorylation and autophagy help drive hepatocyte polarization

Cell polarization requires increased cellular energy and metabolic output, but how these energetic demands are met by polarizing cells is unclear. To address these issues, we investigated the roles of mitochondrial bioenergetics and autophagy during cell polarization of hepatocytes cultured in a collagen sandwich system. We found that as the hepatocytes begin to polarize, they use oxidative phosphorylation to raise their ATP levels, and this energy production is required for polarization. After the cells are polarized, the hepatocytes shift to become more dependent on glycolysis to produce ATP. Along with this central reliance on oxidative phosphorylation as the main source of ATP production in polarizing cultures, several other metabolic processes are reprogrammed during the time course of polarization. As the cells polarize, mitochondria elongate and mitochondrial membrane potential increases. In addition, lipid droplet abundance decreases over time. These findings suggest that polarizing cells are reliant on fatty acid oxidation, which is supported by pharmacologic inhibition of β-oxidation by etomoxir. Finally, autophagy is up-regulated during cell polarization, with inhibition of autophagy retarding cell polarization. Taken together, our results describe a metabolic shift involving a number of coordinated metabolic pathways that ultimately serve to increase energy production during cell polarization.

A new mitochondrial pool of cyclin E, regulated by Drp1, is linked to cell-density-dependent cell proliferation

ABSTRACT The regulation and function of the crucial cell cycle regulator cyclin E (CycE) remains elusive. Unlike other cyclins, CycE can be uniquely controlled by mitochondrial energetics, the exact mechanism being unclear. Using mammalian cells (in vitro) and Drosophila (in vivo) model systems in parallel, we show that CycE can be directly regulated by mitochondria through its recruitment to the organelle. Active mitochondrial bioenergetics maintains a distinct mitochondrial pool of CycE (mtCycE) lacking a key phosphorylation required for its degradation. Loss of the mitochondrial fission protein dynamin-related protein 1 (Drp1, SwissProt O00429 in humans) augments mitochondrial respiration and elevates the mtCycE pool allowing CycE deregulation, cell cycle alterations and enrichment of stem cell markers. Such CycE deregulation after Drp1 loss attenuates cell proliferation in low-cell-density environments. However, in high-cell-density environments, elevated MEK–ERK signaling in the absence of Drp1 releases mtCycE to support escape of contact inhibition and maintain aberrant cell proliferation. Such Drp1-driven regulation of CycE recruitment to mitochondria might be a mechanism to modulate CycE degradation during normal developmental processes as well as in tumorigenic events. Summary: Using mammalian cells and Drosophila, we have identified a new mitochondrial pool of the cell cycle regulator cyclin E that can be modulated by Drp1 to control cell proliferation.