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1.
Sci Rep ; 12(1): 6255, 2022 04 15.
Article in English | MEDLINE | ID: mdl-35428815

ABSTRACT

Bone marrow megakaryocytes (MKs) undergo a maturation involving contacts with the microenvironment before extending proplatelets through sinusoids to deliver platelets in the bloodstream. We demonstrated that MKs assemble linear F-actin-enriched podosomes on collagen I fibers. Microscopy analysis evidenced an inverse correlation between the number of dot-like versus linear podosomes over time. Confocal videomicroscopy confirmed that they derived from each-other. This dynamics was dependent on myosin IIA. Importantly, MKs progenitors expressed the Tks4/5 adaptors, displayed a strong gelatinolytic ability and did not form linear podosomes. While maturing, MKs lost Tks expression together with digestive ability. However, those MKs were still able to remodel the matrix by exerting traction on collagen I fibers through a collaboration between GPVI, ß1 integrin and linear podosomes. Our data demonstrated that a change in structure and composition of podosomes accounted for the shift of function during megakaryopoiesis. These data highlight the fact that members of the invadosome family could correspond to different maturation status of the same entity, to adapt to functional responses required by differentiation stages of the cell that bears them.


Subject(s)
Megakaryocytes , Podosomes , Blood Platelets/metabolism , Collagen Type I/metabolism , Megakaryocytes/metabolism , Thrombopoiesis
2.
EMBO J ; 41(5): e107982, 2022 03 01.
Article in English | MEDLINE | ID: mdl-35178724

ABSTRACT

A paradox of eukaryotic cells is that while some species assemble a complex actin cytoskeleton from a single ortholog, other species utilize a greater diversity of actin isoforms. The physiological consequences of using different actin isoforms, and the molecular mechanisms by which highly conserved actin isoforms are segregated into distinct networks, are poorly known. Here, we sought to understand how a simple biological system, composed of a unique actin and a limited set of actin-binding proteins, reacts to a switch to heterologous actin expression. Using yeast as a model system and biomimetic assays, we show that such perturbation causes drastic reorganization of the actin cytoskeleton. Our results indicate that defective interaction of a heterologous actin for important regulators of actin assembly limits certain actin assembly pathways while reinforcing others. Expression of two heterologous actin variants, each specialized in assembling a different network, rescues cytoskeletal organization and confers resistance to external perturbation. Hence, while species using a unique actin have homeostatic actin networks, actin assembly pathways in species using several actin isoforms may act more independently.


Subject(s)
Actins/metabolism , Protein Isoforms/metabolism , Actin Cytoskeleton/metabolism , Amino Acid Sequence , Microfilament Proteins/metabolism , Saccharomyces cerevisiae/metabolism , Saccharomyces cerevisiae Proteins/metabolism , Sequence Homology, Amino Acid
3.
Nat Commun ; 12(1): 548, 2021 01 22.
Article in English | MEDLINE | ID: mdl-33483497

ABSTRACT

Actin polymerization provides force for vital processes of the eukaryotic cell, but our understanding of actin dynamics and energetics remains limited due to the lack of high-quality probes. Most current probes affect dynamics of actin or its interactions with actin-binding proteins (ABPs), and cannot track the bound nucleotide. Here, we identify a family of highly sensitive fluorescent nucleotide analogues structurally compatible with actin. We demonstrate that these fluorescent nucleotides bind to actin, maintain functional interactions with a number of essential ABPs, are hydrolyzed within actin filaments, and provide energy to power actin-based processes. These probes also enable monitoring actin assembly and nucleotide exchange with single-molecule microscopy and fluorescence anisotropy kinetics, therefore providing robust and highly versatile tools to study actin dynamics and functions of ABPs.


Subject(s)
Actin Cytoskeleton/metabolism , Actins/metabolism , Microfilament Proteins/metabolism , Muscle Proteins/metabolism , Nucleotides/metabolism , Actins/chemistry , Algorithms , Animals , Fluorescence Polarization , Fluorescent Dyes/chemistry , Hydrolysis , Kinetics , Models, Theoretical , Muscle Proteins/chemistry , Nucleotides/chemistry , Protein Binding , Rabbits , Thermodynamics
4.
Open Biol ; 10(9): 200157, 2020 09.
Article in English | MEDLINE | ID: mdl-32873155

ABSTRACT

The actin cytoskeleton has the particularity of being assembled into many functionally distinct filamentous networks from a common reservoir of monomeric actin. Each of these networks has its own geometrical, dynamical and mechanical properties, because they are capable of recruiting specific families of actin-binding proteins (ABPs), while excluding the others. This review discusses our current understanding of the underlying molecular mechanisms that cells have developed over the course of evolution to segregate ABPs to appropriate actin networks. Segregation of ABPs requires the ability to distinguish actin networks as different substrates for ABPs, which is regulated in three different ways: (1) by the geometrical organization of actin filaments within networks, which promotes or inhibits the accumulation of ABPs; (2) by the identity of the networks' filaments, which results from the decoration of actin filaments with additional proteins such as tropomyosin, from the use of different actin isoforms or from covalent modifications of actin; (3) by the existence of collaborative or competitive binding to actin filaments between two or multiple ABPs. This review highlights that all these effects need to be taken into account to understand the proper localization of ABPs in cells, and discusses what remains to be understood in this field of research.


Subject(s)
Actin Cytoskeleton/metabolism , Cytoskeleton/metabolism , Microfilament Proteins/metabolism , Actin Cytoskeleton/chemistry , Actin Cytoskeleton/genetics , Actin Depolymerizing Factors/chemistry , Actin Depolymerizing Factors/metabolism , Amino Acid Sequence , Animals , Biochemical Phenomena , Carrier Proteins/metabolism , Cytoskeleton/chemistry , Humans , Microfilament Proteins/chemistry , Phylogeny , Protein Binding , Protein Isoforms , Protein Processing, Post-Translational , Protein Transport , Tropomyosin/chemistry , Tropomyosin/genetics , Tropomyosin/metabolism
5.
PLoS Biol ; 17(6): e3000317, 2019 06.
Article in English | MEDLINE | ID: mdl-31181075

ABSTRACT

Within the cytoplasm of a single cell, several actin networks can coexist with distinct sizes, geometries, and protein compositions. These actin networks assemble in competition for a limited pool of proteins present in a common cellular environment. To predict how two distinct networks of actin filaments control this balance, the simultaneous assembly of actin-related protein 2/3 (Arp2/3)-branched networks and formin-linear networks of actin filaments around polystyrene microbeads was investigated with a range of actin accessory proteins (profilin, capping protein, actin-depolymerizing factor [ADF]/cofilin, and tropomyosin). Accessory proteins generally affected actin assembly rates for the distinct networks differently. These effects at the scale of individual actin networks were surprisingly not always correlated with corresponding loss-of-function phenotypes in cells. However, our observations agreed with a global interpretation, which compared relative actin assembly rates of individual actin networks. This work supports a general model in which the size of distinct actin networks is determined by their relative capacity to assemble in a common and competing environment.


Subject(s)
Actin-Related Protein 2-3 Complex/metabolism , Actins/metabolism , Microfilament Proteins/physiology , Actin Cytoskeleton/metabolism , Actin Depolymerizing Factors/metabolism , Actin-Related Protein 2-3 Complex/physiology , Animals , Humans , Kinetics , Microfilament Proteins/metabolism , Profilins/metabolism , Protein Interaction Maps/physiology , Tropomyosin
6.
Nat Commun ; 8: 15838, 2017 06 15.
Article in English | MEDLINE | ID: mdl-28643773

ABSTRACT

Blood platelets are produced by large bone marrow (BM) precursor cells, megakaryocytes (MKs), which extend cytoplasmic protrusions (proplatelets) into BM sinusoids. The molecular cues that control MK polarization towards sinusoids and limit transendothelial crossing to proplatelets remain unknown. Here, we show that the small GTPases Cdc42 and RhoA act as a regulatory circuit downstream of the MK-specific mechanoreceptor GPIb to coordinate polarized transendothelial platelet biogenesis. Functional deficiency of either GPIb or Cdc42 impairs transendothelial proplatelet formation. In the absence of RhoA, increased Cdc42 activity and MK hyperpolarization triggers GPIb-dependent transmigration of entire MKs into BM sinusoids. These findings position Cdc42 (go-signal) and RhoA (stop-signal) at the centre of a molecular checkpoint downstream of GPIb that controls transendothelial platelet biogenesis. Our results may open new avenues for the treatment of platelet production disorders and help to explain the thrombocytopenia in patients with Bernard-Soulier syndrome, a bleeding disorder caused by defects in GPIb-IX-V.


Subject(s)
Blood Platelets/enzymology , Platelet Glycoprotein GPIb-IX Complex/metabolism , cdc42 GTP-Binding Protein/metabolism , rhoA GTP-Binding Protein/metabolism , Animals , Blood Platelets/cytology , Cell Polarity , Endothelial Cells/cytology , Endothelial Cells/enzymology , Female , Humans , Megakaryocytes/cytology , Megakaryocytes/enzymology , Mice , Mice, Inbred C57BL , Mice, Knockout , Platelet Glycoprotein GPIb-IX Complex/genetics , cdc42 GTP-Binding Protein/genetics , rhoA GTP-Binding Protein/genetics
7.
Biochimie ; 125: 250-8, 2016 Jun.
Article in English | MEDLINE | ID: mdl-26391221

ABSTRACT

By interacting specifically with proteins, phosphoinositides organize the spatiotemporal formation of protein complexes involved in the control of intracellular signaling, vesicular trafficking and cytoskeleton dynamics. A set of specific kinases and phosphatases ensures the production, degradation and inter-conversion of phosphoinositides to achieve a high level of precision in the regulation of cellular dynamics coordinated by these lipids. The direct involvement of these enzymes in cancer, genetic or infectious diseases, and the recent arrival of inhibitors targeting specific phosphoinositide kinases in clinic, emphasize the importance of these lipids and their metabolism in the biomedical field.


Subject(s)
1-Phosphatidylinositol 4-Kinase/metabolism , Phosphatidylinositols/metabolism , Signal Transduction , 1-Phosphatidylinositol 4-Kinase/genetics , Animals , Genetic Diseases, Inborn/genetics , Genetic Diseases, Inborn/metabolism , Humans , Infections/genetics , Infections/metabolism , Neoplasm Proteins/genetics , Neoplasm Proteins/metabolism , Neoplasms/genetics , Neoplasms/metabolism , Phosphatidylinositols/genetics
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