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Contains fulltext : 282464.pdf (Publisher’s version ) (Open Access)

Contains fulltext : 282464.pdf (Publisher’s version ) (Open Access)

Basement membrane transmigration during embryonal development, tissue homeostasis and tumor invasion relies on invadosomes, a collective term for invadopodia and podosomes. An adequate structural framework for this process is still missing. Here, we reveal the modular actin nano-architecture that enables podosome protrusion and mechanosensing. The podosome protrusive core contains a central branched actin module encased by a linear actin module, each harboring specific actin interactors and actin isoforms. From the core, two actin modules radiate: ventral filaments bound by vinculin and connected to the plasma membrane and dorsal interpodosomal filaments crosslinked by myosin IIA. On stiff substrates, the actin modules mediate long-range substrate exploration, associated with degradative behavior. On compliant substrates, the vinculin-bound ventral actin filaments shorten, resulting in short-range connectivity and a focally protrusive, non-degradative state. Our findings redefine podosome nanoscale architecture and reveal a paradigm for how actin modularity drives invadosome mechanosensing in cells that breach tissue boundaries.

Contains fulltext : 170968.pdf (publisher's version ) (Closed access), Microbial stimuli such as lipopolysaccharide (LPS) induce robust metabolic rewiring in immune cells known as the Warburg effect. It is unknown whether this increase in glycolysis and decrease in oxidative phosphorylation (OXPHOS) is a general characteristic of monocytes that have encountered a pathogen. Using CD14+ monocytes from healthy donors, we demonstrated that most microbial stimuli increased glycolysis, but that only stimulation of Toll-like receptor (TLR) 4 with LPS led to a decrease in OXPHOS. Instead, activation of other TLRs, such as TLR2 activation by Pam3CysSK4 (P3C), increased oxygen consumption and mitochondrial enzyme activity. Transcriptome and metabolome analysis of monocytes stimulated with P3C versus LPS confirmed the divergent metabolic responses between both stimuli, and revealed significant differences in the tricarboxylic acid cycle, OXPHOS and lipid metabolism pathways following stimulation of monocytes with P3C versus LPS. At a functional level, pharmacological inhibition of complex I of the mitochondrial electron transport chain diminished cytokine production and phagocytosis in P3C- but not LPS-stimulated monocytes. Thus, unlike LPS, complex microbial stimuli and the TLR2 ligand P3C induce a specific pattern of metabolic rewiring that involves upregulation of both glycolysis and OXPHOS, which enables activation of host defence mechanisms such as cytokine production and phagocytosis.

Contains fulltext : 170968.pdf (publisher's version ) (Closed access), Microbial stimuli such as lipopolysaccharide (LPS) induce robust metabolic rewiring in immune cells known as the Warburg effect. It is unknown whether this increase in glycolysis and decrease in oxidative phosphorylation (OXPHOS) is a general characteristic of monocytes that have encountered a pathogen. Using CD14+ monocytes from healthy donors, we demonstrated that most microbial stimuli increased glycolysis, but that only stimulation of Toll-like receptor (TLR) 4 with LPS led to a decrease in OXPHOS. Instead, activation of other TLRs, such as TLR2 activation by Pam3CysSK4 (P3C), increased oxygen consumption and mitochondrial enzyme activity. Transcriptome and metabolome analysis of monocytes stimulated with P3C versus LPS confirmed the divergent metabolic responses between both stimuli, and revealed significant differences in the tricarboxylic acid cycle, OXPHOS and lipid metabolism pathways following stimulation of monocytes with P3C versus LPS. At a functional level, pharmacological inhibition of complex I of the mitochondrial electron transport chain diminished cytokine production and phagocytosis in P3C- but not LPS-stimulated monocytes. Thus, unlike LPS, complex microbial stimuli and the TLR2 ligand P3C induce a specific pattern of metabolic rewiring that involves upregulation of both glycolysis and OXPHOS, which enables activation of host defence mechanisms such as cytokine production and phagocytosis.

Contains fulltext : 170968.pdf (publisher's version ) (Closed access), Microbial stimuli such as lipopolysaccharide (LPS) induce robust metabolic rewiring in immune cells known as the Warburg effect. It is unknown whether this increase in glycolysis and decrease in oxidative phosphorylation (OXPHOS) is a general characteristic of monocytes that have encountered a pathogen. Using CD14+ monocytes from healthy donors, we demonstrated that most microbial stimuli increased glycolysis, but that only stimulation of Toll-like receptor (TLR) 4 with LPS led to a decrease in OXPHOS. Instead, activation of other TLRs, such as TLR2 activation by Pam3CysSK4 (P3C), increased oxygen consumption and mitochondrial enzyme activity. Transcriptome and metabolome analysis of monocytes stimulated with P3C versus LPS confirmed the divergent metabolic responses between both stimuli, and revealed significant differences in the tricarboxylic acid cycle, OXPHOS and lipid metabolism pathways following stimulation of monocytes with P3C versus LPS. At a functional level, pharmacological inhibition of complex I of the mitochondrial electron transport chain diminished cytokine production and phagocytosis in P3C- but not LPS-stimulated monocytes. Thus, unlike LPS, complex microbial stimuli and the TLR2 ligand P3C induce a specific pattern of metabolic rewiring that involves upregulation of both glycolysis and OXPHOS, which enables activation of host defence mechanisms such as cytokine production and phagocytosis.

Contains fulltext : 154023.pdf (Publisher’s version ) (Closed access), Subcellular partitioning of creatine kinase contributes to the formation of patterns in intracellular ATP distribution and the fuelling of cellular processes with a high and sudden energy demand. We have previously shown that brain-type creatine kinase (CK-B) accumulates at the phagocytic cup in macrophages where it is involved in the compartmentalized generation of ATP for actin remodeling. Here, we report that CK-B catalytic activity also helps in the formation of protrusive ruffle structures which are actin-dependent and abundant on the surface of both unstimulated and LPS-activated macrophages. Recruitment of CK-B to these structures occurred transiently and inhibition of the enzyme's catalytic activity with cyclocreatine led to a general smoothening of surface morphology as visualized by scanning electron microscopy. Comparison of the dynamics of distribution of YFP-tagged CK-mutants and isoforms by live imaging revealed that amino acid residues in the C-terminal segment (aa positions 323-330) that forms one of the protein's two mobile loops are involved in partitioning over inner regions of the cytosol and nearby sites where membrane protrusions occur during induction of phagocytic cup formation. Although wt CK-B, muscle-type CK (CK-M), and a catalytically dead CK-B-E232Q mutant with intact loop region were normally recruited from the cytosolic pool, no dynamic transition to the phagocytic cup area was seen for the CK-homologue arginine kinase and a CK-B-D326A mutant protein. Bioinformatics analysis helped us to predict that conformational flexibility of the C-terminal loop, independent of conformational changes induced by substrate binding or catalytic activity, is likely involved in exposing the enzyme for binding at or near the sites of membrane protrusion formation.

Contains fulltext : 154023.pdf (Publisher’s version ) (Closed access), Subcellular partitioning of creatine kinase contributes to the formation of patterns in intracellular ATP distribution and the fuelling of cellular processes with a high and sudden energy demand. We have previously shown that brain-type creatine kinase (CK-B) accumulates at the phagocytic cup in macrophages where it is involved in the compartmentalized generation of ATP for actin remodeling. Here, we report that CK-B catalytic activity also helps in the formation of protrusive ruffle structures which are actin-dependent and abundant on the surface of both unstimulated and LPS-activated macrophages. Recruitment of CK-B to these structures occurred transiently and inhibition of the enzyme's catalytic activity with cyclocreatine led to a general smoothening of surface morphology as visualized by scanning electron microscopy. Comparison of the dynamics of distribution of YFP-tagged CK-mutants and isoforms by live imaging revealed that amino acid residues in the C-terminal segment (aa positions 323-330) that forms one of the protein's two mobile loops are involved in partitioning over inner regions of the cytosol and nearby sites where membrane protrusions occur during induction of phagocytic cup formation. Although wt CK-B, muscle-type CK (CK-M), and a catalytically dead CK-B-E232Q mutant with intact loop region were normally recruited from the cytosolic pool, no dynamic transition to the phagocytic cup area was seen for the CK-homologue arginine kinase and a CK-B-D326A mutant protein. Bioinformatics analysis helped us to predict that conformational flexibility of the C-terminal loop, independent of conformational changes induced by substrate binding or catalytic activity, is likely involved in exposing the enzyme for binding at or near the sites of membrane protrusion formation.

Contains fulltext : 154023.pdf (Publisher’s version ) (Closed access), Subcellular partitioning of creatine kinase contributes to the formation of patterns in intracellular ATP distribution and the fuelling of cellular processes with a high and sudden energy demand. We have previously shown that brain-type creatine kinase (CK-B) accumulates at the phagocytic cup in macrophages where it is involved in the compartmentalized generation of ATP for actin remodeling. Here, we report that CK-B catalytic activity also helps in the formation of protrusive ruffle structures which are actin-dependent and abundant on the surface of both unstimulated and LPS-activated macrophages. Recruitment of CK-B to these structures occurred transiently and inhibition of the enzyme's catalytic activity with cyclocreatine led to a general smoothening of surface morphology as visualized by scanning electron microscopy. Comparison of the dynamics of distribution of YFP-tagged CK-mutants and isoforms by live imaging revealed that amino acid residues in the C-terminal segment (aa positions 323-330) that forms one of the protein's two mobile loops are involved in partitioning over inner regions of the cytosol and nearby sites where membrane protrusions occur during induction of phagocytic cup formation. Although wt CK-B, muscle-type CK (CK-M), and a catalytically dead CK-B-E232Q mutant with intact loop region were normally recruited from the cytosolic pool, no dynamic transition to the phagocytic cup area was seen for the CK-homologue arginine kinase and a CK-B-D326A mutant protein. Bioinformatics analysis helped us to predict that conformational flexibility of the C-terminal loop, independent of conformational changes induced by substrate binding or catalytic activity, is likely involved in exposing the enzyme for binding at or near the sites of membrane protrusion formation.