Éditeur(s) : HAL CCSD Landes Bioscience
Résumé : International audience
In 2008 we published the first set of guidelines for standardizing research in autophagy. Since then, research on this topic has continued to accelerate, and many new scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Accordingly, it is important to update these guidelines for monitoring autophagy in different organisms. Various reviews have described the range of assays that have been used for this purpose. Nevertheless, there continues to be confusion regarding acceptable methods to measure autophagy, especially in multicellular eukaryotes. A key point that needs to be emphasized is that there is a difference between measurements that monitor the numbers or volume of autophagic elements (e.g., autophagosomes or autolysosomes) at any stage of the autophagic process vs. those that measure flux through the autophagy pathway (i.e., the complete process); thus, a block in macroautophagy that results in autophagosome accumulation needs to be differentiated from stimuli that result in increased autophagic activity, defined as increased autophagy induction coupled with increased delivery to, and degradation within, lysosomes (in most higher eukaryotes and some protists such as Dictyostelium) or the vacuole (in plants and fungi). In other words, it is especially important that investigators new to the field understand that the appearance of more autophagosomes does not necessarily equate with more autophagy. In fact, in many cases, autophagosomes accumulate because of a block in trafficking to lysosomes without a concomitant change in autophagosome biogenesis, whereas an increase in autolysosomes may reflect a reduction in degradative activity. Here, we present a set of guidelines for the selection and interpretation of methods for use by investigators who aim to examine macroautophagy and related processes, as well as for reviewers who need to provide realistic and reasonable critiques of papers that are focused on these processes. These guidelines are not meant to be a formulaic set of rules, because the appropriate assays depend in part on the question being asked and the system being used. In addition, we emphasize that no individual assay is guaranteed to be the most appropriate one in every situation, and we strongly recommend the use of multiple assays to monitor autophagy. In these guidelines, we consider these various methods of assessing autophagy and what information can, or cannot, be obtained from them. Finally, by discussing the merits and limits of particular autophagy assays, we hope to encourage technical innovation in the field.
Autophagy

hal-00735751
https://hal.archives-ouvertes.fr/hal-00735751
PUBMED : 22966490

Éditeur(s) : HAL CCSD American Society of Hematology
Résumé : International audience
Polycythemia vera (PV) is characterized by an increased RBC mass, spontaneous erythroid colony formation, and the JAK2V617F mutation. PV is associated with a high risk of mesenteric and cerebral thrombosis. PV RBC adhesion to endothelial laminin is increased and mediated by phosphorylated erythroid Lu/BCAM. In the present work, we investigated the mechanism responsible for Lu/BCAM phosphorylation in the presence of JAK2V617F using HEL and BaF3 cell lines as well as RBCs from patients with PV. High levels of Rap1-GTP were found in HEL and BaF3 cells expressing JAK2V617F compared with BaF3 cells with wild-type JAK2. This finding was associated with increased Akt activity, Lu/BCAM phosphorylation, and cell adhesion to laminin that were inhibited by the dominant-negative Rap1S17N or by the specific Rap1 inhibitor GGTI-298. Surprisingly, knocking-down EpoR in HEL cells did not alter Akt activity or cell adhesion to laminin. Our findings reveal a novel EpoR-independent Rap1/Akt signaling pathway that is activated by JAK2V617F in circulating PV RBCs and responsible for Lu/BCAM activation. This new characteristic of JAK2V617F could play a critical role in initiating abnormal interactions among circulating and endothelial cells in patients with PV.
ISSN: 0006-4971

inserm-00821109
http://www.hal.inserm.fr/inserm-00821109
http://www.hal.inserm.fr/inserm-00821109/document
http://www.hal.inserm.fr/inserm-00821109/file/De_Grandis_Blood_2013.pdf
DOI : 10.1182/blood-2012-07-440487

Éditeur(s) : HAL CCSD Ferrata Storti Foundation
Résumé : International audience
BACKGROUND: Abnormal interactions between red blood cells, leukocytes and endothelial cells play a critical role in the occurrence of the painful vaso-occlusive crises associated with sickle cell disease. We investigated the interaction between circulating leukocytes and red blood cells which could lead to aggregate formation, enhancing the incidence of vaso-occlusive crises. DESIGN AND METHODS: Blood samples from patients with sickle cell disease (n=25) and healthy subjects (n=5) were analyzed by imaging and classical flow cytometry after density gradient separation. The identity of the cells in the peripheral blood mononuclear cell layer was determined using antibodies directed specifically against white (anti-CD45) or red (anti-glycophorin A) blood cells. RESULTS: Aggregates between red blood cells and peripheral blood mononuclear cells were visualized in whole blood from patients with sickle cell disease. The aggregation rate was 10-fold higher in these patients than in control subjects. Both mature red blood cells and reticulocytes were involved in these aggregates through their interaction with mononuclear cells, mainly with monocytes. The size of the aggregates was variable, with one mononuclear cell binding to one, two or several red blood cells. Erythroid Lu/basal cell adhesion molecule and α(4)β(1) integrin were involved in aggregate formation. The aggregation rate was lower in patients treated with hydroxycarbamide than in untreated patients. CONCLUSIONS: Our study gives visual evidence of the existence of circulating red blood cell-peripheral blood mononuclear cell aggregates in patients with sickle cell disease and shows that these aggregates are decreased during hydroxycarbamide treatment. Our results strongly suggest that erythroid Lu/basal cell adhesion molecule proteins are implicated in these aggregates through their interaction with α(4)β(1) integrin on peripheral blood mononuclear cells.
ISSN: 0390-6078

inserm-00821120
http://www.hal.inserm.fr/inserm-00821120
http://www.hal.inserm.fr/inserm-00821120/document
http://www.hal.inserm.fr/inserm-00821120/file/1841.full.pdf
DOI : 10.3324/haematol.2010.026294

Éditeur(s) : HAL CCSD Portland Press
Résumé : International audience
Lutheran (Lu) blood group and Basal Cell Adhesion Molecule (BCAM) antigens both reside on two glycoprotein (gp) isoforms, Lu and Lu(v13), that differ by the size of their cytoplasmic tail. They are receptors of laminin-10/11 and are expressed in red blood cells (RBCs), epithelial cells of multiple tissues and vascular endothelial cells. To gain more insights into the biological function of Lu/BCAM gps, we looked for potential partners of their cytoplasmic tail. We isolated Ubc9 (Ubiquitin-conjugating enzyme 9) protein by screening a human kidney library using the yeast two-hybrid system. Lu/Ubc9 interaction was validated by GST pull-down and co-immunoprecipitation experiments. Endogenous Ubc9 formed a complex with endogenous or recombinant Lu gp in A498 and MDCK epithelial cells, respectively. Substitution of lysine 585 by alanine in the Lu gp, abolished in vitro and ex vivo interaction of Lu gp with Ubc9 protein. Lu K585A mutant transfected in MDCK cells exhibited a normal basolateral membrane expression but was overexpressed at the surface of polarized MDCK cells as compared to wild-type Lu. Pulse-chase experiments showed extended half-life of Lu K585A gp at the plasma membrane, suggesting an impaired endocytosis of this mutant leading to protein accumulation at the membrane. Furthermore, we showed that the ability of MDCK-Lu K585A cells to spread on immobilized laminin was dramatically decreased. Our data supports a physiological role for the direct interaction between Lu gp and Ubc9 protein, and reveals a role for this enzyme in regulating the stability of Lu gp at the cell membrane.
ISSN: 0264-6021

hal-00478614
https://hal.archives-ouvertes.fr/hal-00478614
https://hal.archives-ouvertes.fr/hal-00478614/document
https://hal.archives-ouvertes.fr/hal-00478614/file/PEER_stage2_10.1042%252FBJ20060861.pdf
DOI : 10.1042/BJ20060861

Éditeur(s) : HAL CCSD Elsevier
Résumé : The Duffy antigen/receptor for chemokine (DARC) is an erythrocyte receptor for malaria parasites (Plasmodium vivax and Plasmodium knowlesi) and for chemokines. In contrast to other chemokine receptors, DARC is a promiscuous receptor that binds chemokines of both CC and CXC classes. The four extracellular domains (ECDs) of DARC are essential for its interaction with chemokines, whilst the first (ECD1) is sufficient for the interaction with malaria erythrocyte-binding protein. In this study, we elaborate and analyze structural models of the DARC. The construction of the 3D models is based on a comparative modeling process and on the use of many procedures to predict transmembrane segments and to detect far homologous proteins with known structures. Threading, ab initio, secondary structure and Protein Blocks approaches are used to build a very large number of models. The conformational exploration of the ECDs is performed with simulated annealing. The second and fourth ECDs are strongly constrained. On the contrary, the ECD1 is highly flexible, but seems composed of three consecutive regions: a small beta-sheet, a linker region and a structured loop. The chosen structural models encompass most of the biochemical features and reflect the known experimental data. They may be used to analyze functional interaction properties.
ISSN: 0006-3002

inserm-00143373
http://www.hal.inserm.fr/inserm-00143373
http://www.hal.inserm.fr/inserm-00143373/document
http://www.hal.inserm.fr/inserm-00143373/file/de_Brevern_BBA_2005.pdf
DOI : 10.1016/j.bbagen.2005.05.016

Éditeur(s) : HAL CCSD Wiley
Résumé : International audience
ISSN: 0007-1048

hal-01275163
https://hal.archives-ouvertes.fr/hal-01275163
DOI : 10.1111/bjh.13937

Éditeur(s) : HAL CCSD American Physiological Society
Résumé : International audience
Zidi-Yahiaoui N, Callebaut I, Genetet S, Le Van Kim C, Cartron JP, Colin Y, Ripoche P, Mouro-Chanteloup I. Functional analysis of human RhCG: comparison with E. coli ammonium transporter reveals similarities in the pore and differences in the vestibule. Am J Physiol Cell Physiol 297: C537–C547, 2009. First published June 24, 2009; doi:10.1152/ajpcell.00137.2009.—Rh glycoproteins are members of the ammonium transporter (Amt)/methylamine permease (Mep)/Rh family facilitating movement of NH3 across plasma membranes. Homology models constructed on the basis of the experimental structures of Escherichia coli AmtB and Nitrosomonas europaea Rh50 indicated a channel structure for human RhA (RhAG), RhB (RhBG), and RhC (RhCG) glycoproteins in which external and internal vestibules are linked by a pore containing two strictly conserved histidines. The pore entry is constricted by two highly conserved phenylalanines, “twin-Phe.” In this study, RhCG function was investigated by stopped-flow spectrofluorometry measuring kinetic pH variations in HEK293E cells in the presence of an ammonium gradient. The apparent unitary NH3 permeability of RhCG was determined and was found to be close to that of AmtB. With a site-directed mutagenesis approach, critical residues involved in Rh NH3 channel activity were highlighted. In the external vestibule, the importance of both the charge and the conformation of the conserved aspartic acid was shown. In contrast to AmtB, individual mutations of each phenylalanine of the twin-Phe impaired the function while the removal of both resulted in recovery of the transport activity. The impact of the mutations suggests that, although having a common function and a similar channel structure, bacterial AmtB and human Rh vary in several aspects of the NH3 transport mechanisms.
ISSN: 0363-6143

hal-00417492
https://hal.archives-ouvertes.fr/hal-00417492
DOI : 10.1152/ajpcell.00137.2009.

Éditeur(s) : HAL CCSD Wiley-Blackwell
Résumé : International audience
American Journal of Hematology

hal-01113050
https://hal.archives-ouvertes.fr/hal-01113050
DOI : 10.1002/ajh.23941

Éditeur(s) : HAL CCSD American Society of Hematology
Résumé : International audience
Sickle cell disease is characterized by painful vaso-occlusive crises during which abnormal interactions between erythroid adhesion molecules and vessel-wall proteins are thought to play a critical role. Hydroxyurea, the only drug with proven benefit in sickle cell disease, diminishes these interactions, but its mechanism of action is not fully understood. We report that, under hydroxyurea, expression of the unique erythroid laminin receptor Lu/BCAM was increased, but red blood cell adhesion to laminin decreased. Because Lu/BCAM phosphorylation is known to activate cell adhesion to laminin, it was evaluated and found to be dramatically lower in hydroxyurea-treated patients. Analysis of the protein kinase A pathway showed decreased intracellular levels of the upstream effector cyclic adenosine monophosphate during hydroxyurea treatment. Using a cellular model expressing recombinant Lu/BCAM, we showed that hydroxyurea led to decreased intracellular cyclic adenosine monophosphate levels and diminished Lu/BCAM phosphorylation and cell adhesion. We provide evidence that hydroxyurea could reduce abnormal sickle red blood cell adhesion to the vascular wall by regulating the activation state of adhesion molecules independently of their expression level.
ISSN: 0006-4971

inserm-00821117
http://www.hal.inserm.fr/inserm-00821117
http://www.hal.inserm.fr/inserm-00821117/document
http://www.hal.inserm.fr/inserm-00821117/file/Bartolucci_Blood_2010.pdf
DOI : 10.1182/blood-2009-12-257444

Éditeur(s) : HAL CCSD
Résumé : International audience
In this paper we present a microfluidic architecture dedicated to the study of Red Blood Cells (RBCs) deformability in microchannel smaller than the cell shape (biconcave disc of 7-8 μm in diameter) and cell capability to pass through slits separated by a gap value down to 2 μm, acting as a filter. This device is use in the context of RBCs disorders affecting their deformability, which is analysed versus number of cells trapped in the filter. This device can be seen as mimicking the human spleen. Microfabrication of the chip is based on polydimethylsiloxane (PDMS) associated to a two level thick photoresist mould (SU8). One level is used for microfluidic access with a 25 μm thickness towards the studied zone. The second level concerns the mimicking zones where RBCs flow through 5 μm down to 2 μm-wide slits acting like a mechanical filter. This level has a thickness of 5 μm. Devices have been tested on RBCs having specific disorder on their mechanical elasticity in order to validate the design principle. Two cases are presented in this paper: a.) Sickle RBCs inducing cell rigidity at two levels b.) Parasitized RBCs by Plasmodium falciparum. On both cases, we clearly show that deformability of RBCs influences the capability to pass through small slits (down to 2um) and thus is in relation with the pathology stage. This spleen-like microfluidic chip mimicking the RBCs filtering function is a promising tool for the exploration of RBC disorders combined with mechanical alterations.
4th European Conference on Microfluidics
Limerick, Ireland

hal-01113061
https://hal.archives-ouvertes.fr/hal-01113061