A proteomic-informed view of the changes induced by loss of cellular adherence: The example of mouse macrophages

Except cells circulating in the bloodstream, most cells in vertebrates are adherent. Studying the repercussions of adherence per se in cell physiology is thus very difficult to carry out, although it plays an important role in cancer biology, e.g. in the metastasis process. In order to study how adherence impacts major cell functions, we used a murine macrophage cell line. Opposite to the monocyte/macrophage system, where adherence is associated with the acquisition of differentiated functions, these cells can be grown in both adherent or suspension conditions without altering their differentiated functions (phagocytosis and inflammation signaling). We used a proteomic approach to cover a large panel of proteins potentially modified by the adherence status. Targeted experiments were carried out to validate the proteomic results, e.g. on metabolic enzymes, mitochondrial and cytoskeletal proteins. The mitochondrial activity was increased in non-adherent cells compared with adherent cells, without differences in glucose consumption. Concerning the cytoskeleton, a rearrangement of the actin organization (filopodia vs sub-cortical network) and of the microtubule network were observed between adherent and non-adherent cells. Taken together, these data show the mechanisms at play for the modification of the cytoskeleton and also modifications of the metabolic activity between adherent and non-adherent cells.

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Proteomic profiling of mitochondrial-derived vesicles in brain reveals enrichment of respiratory complex sub-assemblies and small TIM chaperones

10.1101/2020.07.06.189993 ◽

2020 ◽

Author(s):

Rosalind F. Roberts ◽

Andrew N. Bayne ◽

Thomas Goiran ◽

Dominique Lévesque ◽

François-Michel Boisvert ◽

...

Keyword(s):

Protein Transport ◽

Disease Genes ◽

Proteomic Profiling ◽

Cell Functions ◽

Peroxisomal Biogenesis ◽

Proteomic Approach ◽

Significant Difference ◽

Vital Cell ◽

The Brain

ABSTRACTThe generation of mitochondrial-derived vesicles (MDVs) is implicated in a plethora of vital cell functions, from mitochondrial quality control to peroxisomal biogenesis. The discovery of distinct subtypes of MDVs has revealed the selective inclusion of mitochondrial cargo in response to varying stimuli. However, the true scope and variety of MDVs is currently unclear, and unbiased approaches have yet to be used to understand their biology. Furthermore, as mitochondrial dysfunction has been implicated in many neurodegenerative diseases, it is essential to understand MDV pathways in the nervous system. To address this, we sought to identify the cargo in brain MDVs. We used an in vitro budding assay and proteomic approach to identify proteins selectively enriched in MDVs. 72 proteins were identified as MDV enriched, of which 31% were OXPHOS proteins. Interestingly, the OXPHOS proteins localized to specific modules of the respiratory complexes, hinting at the inclusion of sub-assemblies in MDVs. Small TIM chaperones were also highly enriched in MDVs, linking mitochondrial chaperone-mediated protein transport to MDV formation. As the two Parkinson’s disease genes PINK1 and Parkin have been previously implicated in MDV biogenesis in response to oxidative stress, we compared the MDV proteomes from the brains of wild-type mice with those of PINK1-/- and Parkin-/- mice. No significant difference was found, suggesting that PINK1- and Parkin-dependent MDVs make up a small proportion of all MDVs in the brain. Our findings demonstrate a previously uncovered landscape of MDV complexity and provide a foundation from which to discover further novel MDV functions.

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Central metabolism of functionally heterogeneous mesenchymal stromal cells

Scientific Reports ◽

10.1038/s41598-019-51937-9 ◽

2019 ◽

Vol 9 (1) ◽

Cited By ~ 3

Author(s):

Mario Barilani ◽

Roberta Palorini ◽

Giuseppina Votta ◽

Roberta Piras ◽

Giuseppe Buono ◽

...

Keyword(s):

Stem Cells ◽

Oxidative Phosphorylation ◽

Cell Fate ◽

Lactate Production ◽

Glucose Consumption ◽

Mitochondrial Activity ◽

Central Metabolism ◽

Mt Dna ◽

Differentiation Potential ◽

And Function

Abstract Metabolism and mitochondrial biology have gained a prominent role as determinants of stem cell fate and function. In the context of regenerative medicine, innovative parameters predictive of therapeutic efficacy could be drawn from the association of metabolic or mitochondrial parameters to different degrees of stemness and differentiation potentials. Herein, this possibility was addressed in human mesenchymal stromal/stem cells (hMSC) previously shown to differ in lifespan and telomere length. First, these hMSC were shown to possess significantly distinct proliferation rate, senescence status and differentiation capacity. More potential hMSC were associated to higher mitochondrial (mt) DNA copy number and lower mtDNA methylation. In addition, they showed higher expression levels of oxidative phosphorylation subunits. Consistently, they exhibited higher coupled oxygen consumption rate and lower transcription of glycolysis-related genes, glucose consumption and lactate production. All these data pointed at oxidative phosphorylation-based central metabolism as a feature of higher stemness-associated hMSC phenotypes. Consistently, reduction of mitochondrial activity by complex I and III inhibitors in higher stemness-associated hMSC triggered senescence. Finally, functionally higher stemness-associated hMSC showed metabolic plasticity when challenged by glucose or glutamine shortage, which mimic bioenergetics switches that hMSC must undergo after transplantation or during self-renewal and differentiation. Altogether, these results hint at metabolic and mitochondrial parameters that could be implemented to identify stem cells endowed with superior growth and differentiation potential.

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On the Role of Calpain and Rho Proteins in Regulating Integrin-induced Signaling

Thrombosis and Haemostasis ◽

10.1055/s-0037-1615857 ◽

1999 ◽

Vol 82 (08) ◽

pp. 385-391 ◽

Cited By ~ 29

Author(s):

Joan Fox

Keyword(s):

Signaling Molecules ◽

Cytoskeletal Proteins ◽

Adherent Cells ◽

Rho Proteins ◽

Ligand Interactions ◽

Platelet Aggregate ◽

And Migration ◽

Key Steps ◽

A Site ◽

Induced Changes

SummaryThe integrin family of transmembrane receptors plays an essential role in inducing the adhesion of cells to the extracellular matrix. In some cases, members of this family of receptors can bind soluble ligands or can bind receptors on other cells and, in this way, mediate interactions between cells. In all cases, once an integrin has bound, ligand signals are transmitted across the occupied integrin. These signals culminate in changes in the behavior of the cell appropriate for the adherent state of the cell. For example, in the case of platelets, an end result of the signaling induced by binding of fibrinogen to αIIbβ3 in a platelet aggregate is a reorganization of the cytoskeleton that leads to retraction of externally-bound fibrin by clots.1,2 In the case of neutrophils, cytoskeletal changes following the integrininduced interaction of neutrophils with endothelial cells lead to the migration of neutrophils into a site of injury.3,4 Other examples of the consequences of integrin-induced signaling in adherent cells include the trafficking of lymphocytes and migration of cells during development, angiogenesis, and metastasis.5-7 Numerous signaling molecules have been shown to be activated following integrin-ligand interactions.8 Many of these associate in complexes with ligand-occupied integrin and cytoskeletal proteins. However, in general, little is known about the key steps involved regarding integrin-induced changes in the behavior of adherent cells. The present chapter reviews steps involved in integrin-induced signaling, describes the evidence that calpain is one of the signaling molecules involved in this signal transduction, and discusses potential mechanisms by which cleavage of cytoskeletal proteins and signaling molecules by calpain may regulate the integrin-induced changes in cell behavior.

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The α6β4 integrin promotes resistance to ferroptosis

The Journal of Cell Biology ◽

10.1083/jcb.201701136 ◽

2017 ◽

Vol 216 (12) ◽

pp. 4287-4297 ◽

Cited By ~ 29

Author(s):

Caitlin W. Brown ◽

John J. Amante ◽

Hira Lal Goel ◽

Arthur M. Mercurio

Keyword(s):

Lipid Peroxidation ◽

Fatty Acids ◽

Extracellular Matrix ◽

Cancer Biology ◽

Membrane Lipids ◽

Lipid Peroxides ◽

Causal Link ◽

Carcinoma Cells ◽

Adherent Cells ◽

Α6β4 Integrin

Increases in lipid peroxidation can cause ferroptosis, a form of cell death triggered by inhibition of glutathione peroxidase 4 (GPX4), which catalyzes the reduction of lipid peroxides and is a target of ferroptosis inducers, such as erastin. The α6β4 integrin protects adherent epithelial and carcinoma cells from ferroptosis induced by erastin. In addition, extracellular matrix (ECM) detachment is a physiologic trigger of ferroptosis, which is evaded by α6β4. The mechanism that enables α6β4 to evade ferroptosis involves its ability to protect changes in membrane lipids that are proferroptotic. Specifically, α6β4-mediated activation of Src and STAT3 suppresses expression of ACSL4, an enzyme that enriches membranes with long polyunsaturated fatty acids and is required for ferroptosis. Adherent cells lacking α6β4 require an inducer, such as erastin, to undergo ferroptosis because they sustain GPX4 expression, despite their increase in ACSL4. In contrast, ECM detachment of cells lacking α6β4 is sufficient to trigger ferroptosis because GPX4 is suppressed. This causal link between α6β4 and ferroptosis has implications for cancer biology and therapy.

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Heteromeric amino acid transporters: biochemistry, genetics, and physiology

AJP Renal Physiology ◽

10.1152/ajprenal.2001.281.6.f995 ◽

2001 ◽

Vol 281 (6) ◽

pp. F995-F1018 ◽

Cited By ~ 120

Author(s):

Josep Chillarón ◽

Ramón Roca ◽

Alfonso Valencia ◽

Antonio Zorzano ◽

Manuel Palacín

Keyword(s):

Amino Acid ◽

Amino Acid Transport ◽

Human Genetics ◽

Disulfide Bridge ◽

Transport Systems ◽

Cell Physiology ◽

Amino Acid Transporters ◽

Acid Transport ◽

Membrane Glycoproteins ◽

Cell Functions

The heteromeric amino acid transporters (HATs) are composed of two polypeptides: a heavy subunit (HSHAT) and a light subunit (LSHAT) linked by a disulfide bridge. HSHATs are N-glycosylated type II membrane glycoproteins, whereas LSHATs are nonglycosylated polytopic membrane proteins. The HSHATs have been known since 1992, and the LSHATs have been described in the last three years. HATs represent several of the classic mammalian amino acid transport systems (e.g., L isoforms, y+L isoforms, asc, x[Formula: see text], and b0,+). Members of the HAT family are the molecular bases of inherited primary aminoacidurias cystinuria and lysinuric protein intolerance. In addition to the role in amino acid transport, one HSHAT [the heavy subunit of the cell-surface antigen 4F2 (also named CD98)] is involved in other cell functions that might be related to integrin activation. This review covers the biochemistry, human genetics, and cell physiology of HATs, including the multifunctional character of CD98.

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Bioenergetics and mitochondrial transmembrane potential during differentiation of cultured osteoblasts

AJP Cell Physiology ◽

10.1152/ajpcell.2000.279.4.c1220 ◽

2000 ◽

Vol 279 (4) ◽

pp. C1220-C1229 ◽

Cited By ~ 115

Author(s):

Svetlana V. Komarova ◽

Fasoil I. Ataullakhanov ◽

Ruth K. Globus

Keyword(s):

Transmembrane Potential ◽

Lactate Production ◽

Glucose Consumption ◽

Confocal Imaging ◽

Metabolic Inhibitors ◽

Mitochondrial Activity ◽

Atp Production ◽

Fourfold Increase ◽

Fetal Rat ◽

Immature Cells

To evaluate the relationship between osteoblast differentiation and bioenergetics, cultured primary osteoblasts from fetal rat calvaria were grown in medium supplemented with ascorbate to induce differentiation. Before ascorbate treatment, the rate of glucose consumption was 320 nmol · h−1· 106cells−1, respiration was 40 nmol · h−1· 106cells−1, and the ratio of lactate production to glucose consumption was ∼2, indicating that glycolysis was the main energy source for immature osteoblasts. Ascorbate treatment for 14 days led to a fourfold increase in respiration, a threefold increase in ATP production, and a fivefold increase in ATP content compared with that shown in immature cells. Confocal imaging of mitochondria stained with a transmembrane potential-sensitive vital dye showed that mature cells possessed abundant amounts of high-transmembrane-potential mitochondria, which were concentrated near the culture medium-facing surface. Acute treatment of mature osteoblasts with metabolic inhibitors showed that the rate of glycolysis rose to maintain the cellular energy supply constant. Thus progressive differentiation coincided with changes in cellular metabolism and mitochondrial activity, which are likely to play key roles in osteoblast function.

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010.Coordinating the transition from egg to embryo in mammals

Reproduction Fertility and Development ◽

10.1071/srb04abs010 ◽

2004 ◽

Vol 16 (9) ◽

pp. 10 ◽

Cited By ~ 1

Author(s):

J. Carroll

Keyword(s):

Tca Cycle ◽

Cell Types ◽

Mitotic Division ◽

Cortical Granule ◽

Mitochondrial Activity ◽

Mammalian Development ◽

Atp Production ◽

Downstream Target ◽

Cell Functions ◽

Cortical Granule Exocytosis

At fertilization of mammalian oocytes, the sperm induces a series of increases in the concentration of intracellular Ca2+. These Ca2+ oscillations trigger all the events of egg activation, including cortical granule exocytosis, completion of meiosis and entry into the first mitotic division. Thus, intracellular Ca2+ plays a pivotal role in coordinating the transition from egg to embryo. Our work is focussed on understanding how the oocyte prepares for fertilisation, how the Ca2+ oscillations are controlled and how Ca2+ stimulates signalling pathways that lead to optimal early embryonic development. In this lecture I will focus on the downstream pathways of Ca2+ signalling at fertilisation. Conventional Protein Kinase C (cPKC) is the major downstream target of Ca2+ in many cell functions. Using PKC-GFP fusion proteins we have found that cPKC is recruited to the membrane in a manner that is dependent on the frequency and amplitude of the Ca2+ oscillations. Recruitment of cPKC appears to promote the Ca2+ influx necessary to sustain the generation of long lasting Ca2+ oscillations. In other cell types cytosolic Ca2+ increases are known to stimulate mitochondrial respiration. We have found that maintenance of resting Ca2+ levels and sperm-induced Ca2+ oscillations are critically dependent on mitochondrial ATP production: a feature not shared by many cell types. Since Ca2+ release increases ATP consumption we investigated whether the Ca2+ transients increase mitochondrial activity so as to meet this increase in demand. Monitoring autofluorescence from NADH and flavoproteins reveals that Ca2+ transients stimulate a change in redox state of mitochondria, presumably by activating Ca2+-sensitive dehydrogenases of the TCA cycle. Thus, through activation of downstream pathways, including PKC, cyclin B degradation and mitochondrial activity, intracellular Ca2+ provides a signal that orchestrates the activation of early mammalian development.

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Octadecaneuropeptide (ODN) Induces N2a Cells Differentiation through a PKA/PLC/PKC/MEK/ERK-Dependent Pathway: Incidence on Peroxisome, Mitochondria, and Lipid Profiles

Molecules ◽

10.3390/molecules24183310 ◽

2019 ◽

Vol 24 (18) ◽

pp. 3310 ◽

Cited By ~ 7

Author(s):

Namsi ◽

Nury ◽

Khan ◽

Leprince ◽

Vaudry ◽

...

Keyword(s):

Neurodegenerative Diseases ◽

Cell Viability ◽

Neuronal Differentiation ◽

Nerve Cell ◽

Cell Damage ◽

Mitochondrial Activity ◽

Cell Functions ◽

N2a Cells ◽

Topographical Distribution ◽

The Impact

Neurodegenerative diseases are characterized by oxidative stress, mitochondrial damage, and death of neuronal cells. To counteract such damage and to favor neurogenesis, neurotrophic factors could be used as therapeutic agents. Octadecaneuropeptide (ODN), produced by astrocytes, is a potent neuroprotective agent. In N2a cells, we studied the ability of ODN to promote neuronal differentiation. This parameter was evaluated by phase contrast microscopy, staining with crystal violet, cresyl blue, and Sulforhodamine 101. The effect of ODN on cell viability and mitochondrial activity was determined with fluorescein diacetate and DiOC6(3), respectively. The impact of ODN on the topography of mitochondria and peroxisomes, two tightly connected organelles involved in nerve cell functions and lipid metabolism, was evaluated by transmission electron microscopy and fluorescence microscopy: detection of mitochondria with MitoTracker Red, and peroxisome with an antibody directed against the ABCD3 peroxisomal transporter. The profiles in fatty acids, cholesterol, and cholesterol precursors were determined by gas chromatography, in some cases coupled with mass spectrometry. Treatment of N2a cells with ODN (10−14 M, 48 h) induces neurite outgrowth. ODN-induced neuronal differentiation was associated with modification of topographical distribution of mitochondria and peroxisomes throughout the neurites and did not affect cell viability and mitochondrial activity. The inhibition of ODN-induced N2a differentiation with H89, U73122, chelerythrine and U0126 supports the activation of a PKA/PLC/PKC/MEK/ERK-dependent signaling pathway. Although there is no difference in fatty acid profile between control and ODN-treated cells, the level of cholesterol and some of its precursors (lanosterol, desmosterol, lathosterol) was increased in ODN-treated cells. The ability of ODN to induce neuronal differentiation without cytotoxicity reinforces the interest for this neuropeptide with neurotrophic properties to overcome nerve cell damage in major neurodegenerative diseases.

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Testis-Specific Isoform of Na/K-ATPase (ATP1A4) Interactome in Raft and Non-Raft Membrane Fractions from Capacitated Bovine Sperm

International Journal of Molecular Sciences ◽

10.3390/ijms20133159 ◽

2019 ◽

Vol 20 (13) ◽

pp. 3159 ◽

Cited By ~ 2

Author(s):

Gayathri D. Rajamanickam ◽

John P. Kastelic ◽

Jacob C. Thundathil

Keyword(s):

Plasma Membrane ◽

Growth Factor Receptor ◽

Cytoskeletal Proteins ◽

Histone H4 ◽

Glutathione S Transferase ◽

Bovine Sperm ◽

Proteomic Approach ◽

Extracellular Signal ◽

Transport Carrier ◽

Raft Domains

The plasma membrane of sperm contains highly dynamic lipid microdomains (rafts), which house signaling proteins with a role in regulating capacitation. We reported that ATP1A4, the testis-specific isoform of Na/K-ATPase, interacted with caveolin-1, Src, epidermal growth factor receptor (EGFR) and extracellular signal-regulated kinases 1/2 (ERK1/2) in raft and non-raft domains of the plasma membrane of bovine sperm during capacitation. The objective of the present study was to use a proteomic approach to characterize the ATP1A4 interactome in rafts and non-rafts from capacitated bovine sperm. The non-raft interactome included hexokinase 1, plakophilin 1, desmoglein 1, 14-3-3 protein ζ/δ, cathepsin D and heat shock protein beta1 proteins exclusively, whereas glutathione S-transferase and annexin A2 were unique to raft interactome. However, a disintegrin and metalloprotease 32 (ADAM 32), histone H4, actin, acrosin, serum albumin and plakoglobin were identified in both raft and non-raft fractions of capacitated sperm. Based on gene ontology studies, these differentially interacted proteins were implicated in cell–cell adhesion, signal transduction, fertilization, metabolism, proteolysis and DNA replication, in addition to acting as transport/carrier and cytoskeletal proteins. Overall, we identified proteins not previously reported to interact with ATP1A4; furthermore, we inferred that ATP1A4 may have a role in sperm capacitation.

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Gcn5p and Ubp8p Affect Protein Ubiquitylation and Cell Proliferation by Altering the Fermentative/Respiratory Flux Balance in Saccharomyces cerevisiae

mBio ◽

10.1128/mbio.01504-20 ◽

2020 ◽

Vol 11 (4) ◽

Author(s):

Antonella De Palma ◽

Giulia Fanelli ◽

Elisabetta Cretella ◽

Veronica De Luca ◽

Raffaele Antonio Palladino ◽

...

Keyword(s):

Saccharomyces Cerevisiae ◽

Glucose Consumption ◽

Redox Balance ◽

Glycolytic Enzymes ◽

Glycolytic Flux ◽

Direct Role ◽

Content Type ◽

Proteomic Approach ◽

Altered Glucose

ABSTRACT Protein ubiquitylation regulates not only endocellular trafficking and proteasomal degradation but also the catalytic activity of enzymes. In Saccharomyces cerevisiae, we analyzed the composition of the ubiquitylated proteomes in strains lacking acetyltransferase Gcn5p, Ub-protease Ubp8p, or both to understand their involvement in the regulation of protein ubiquitylation. We analyzed His6Ub proteins with a proteomic approach coupling micro-liquid chromatography and tandem mass spectrometry (μLC-MS/MS) in gcn5Δ, ubp8Δ and ubp8Δ gcn5Δ strains. The Ub-proteome altered in the absence of Gcn5p, Ubp8p, or both was characterized, showing that 43% of the proteins was shared in all strains, suggesting their functional relationship. Remarkably, all major glycolytic enzymes showed increased ubiquitylation. Phosphofructokinase 1, the key enzyme of glycolytic flux, showed a higher and altered pattern of ubiquitylation in gcn5Δ and ubp8Δ strains. Severe defects of growth in poor sugar and altered glucose consumption confirmed a direct role of Gcn5p and Ubp8p in affecting the REDOX balance of the cell. IMPORTANCE We propose a study showing a novel role of Gcn5p and Ubp8p in the process of ubiquitylation of the yeast proteome which includes main glycolytic enzymes. Interestingly, in the absence of Gcn5p and Ubp8p glucose consumption and redox balance were altered in yeast. We believe that these results and the role of Gcn5p and Ubp8p in sugar metabolism might open new perspectives of research leading to novel protocols for counteracting the enhanced glycolysis in tumors.

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