Super-resolution fluorescence imaging of membrane nanoscale architectures of hematopoietic stem cell homing and migration molecules

Handle URI:
http://hdl.handle.net/10754/626333
Title:
Super-resolution fluorescence imaging of membrane nanoscale architectures of hematopoietic stem cell homing and migration molecules
Authors:
AbuZineh, Karmen ( 0000-0003-1459-1422 )
Abstract:
Recent development of super-resolution (SR) fluorescence microscopy techniques has provided a new tool for direct visualization of subcellular structures and their dynamics in cells. The homing of Hematopoietic stem/progenitor cells (HSPCs) to bone marrow is a multistep process that is initiated by tethering of HSPCs to endothelium and mediated by spatiotemporally organised ligand-receptor interactions of selectins expressed on endothelial cells to their ligands expressed on HSPCs which occurs against the shear stress exerted by blood flow. Although molecules and biological processes involved in this multi-step cellular interaction have been studied extensively, molecular mechanisms of the homing, in particular the nanoscale spatiotemporal behaviour of ligand-receptor interactions and their role in the cellular interaction, remain elusive. Using our new method of microfluidics-based super-resolution fluorescence imaging platform we can now characterize the correlation between both nanoscale ligand-receptor interactions and tethering/rolling of cells under external shear stress. We found that cell rolling on E-selectin caused significant reorganization of the nanoscale clustering behavior of CD44 and CD43, from a patchy clusters of ~ 200 nm in size to an elongated network-like structures where for PSGL-1 the clustering size did not change significantly as it was 85 nm and after cell rolling the PSGL-1 aggregated to one side or even exhibited an increase in the footprint. Furthermore, I have established the use of 3D SR images that indicated that the patchy clusters of CD44 localize to protruding structures of the cell surface. On the other hand, a significant amount of the network-like elongated CD44 clusters observed after the rolling were located in the close proximity to the E-selectin surface. The effect of the nanoscale reorganization of the clusters on the HSPC rolling over selectins is still an open question at this stage. Nevertheless, my results further demonstrate that this mechanical force-induced reorganisation is accompanied by a large structural reorganisation of actin cytoskeleton. Our microfluidics-based SR imaging also demonstrate an essential role of the nanoscale clustering of CD44 on stable rolling behaviours of cells. Our new experimental platform enhances understanding of the relationship between nanoscopic ligand-receptor interactions and macroscopic cellular interactions, providing a foundation for characterizing complicated HSPC homing
Advisors:
Habuchi, Satoshi ( 0000-0002-6663-2807 )
Committee Member:
Fabrizio, Enzo Di ( 0000-0001-5886-4678 ) ; Merzaban, Jasmeen ( 0000-0002-7276-2907 ) ; Dedecker, Peter
KAUST Department:
Biological and Environmental Sciences and Engineering (BESE) Division
Program:
Bioscience
Issue Date:
Dec-2017
Type:
Dissertation
Appears in Collections:
Dissertations

Full metadata record

DC FieldValue Language
dc.contributor.advisorHabuchi, Satoshien
dc.contributor.authorAbuZineh, Karmenen
dc.date.accessioned2017-12-10T08:40:17Z-
dc.date.available2017-12-10T08:40:17Z-
dc.date.issued2017-12-
dc.identifier.urihttp://hdl.handle.net/10754/626333-
dc.description.abstractRecent development of super-resolution (SR) fluorescence microscopy techniques has provided a new tool for direct visualization of subcellular structures and their dynamics in cells. The homing of Hematopoietic stem/progenitor cells (HSPCs) to bone marrow is a multistep process that is initiated by tethering of HSPCs to endothelium and mediated by spatiotemporally organised ligand-receptor interactions of selectins expressed on endothelial cells to their ligands expressed on HSPCs which occurs against the shear stress exerted by blood flow. Although molecules and biological processes involved in this multi-step cellular interaction have been studied extensively, molecular mechanisms of the homing, in particular the nanoscale spatiotemporal behaviour of ligand-receptor interactions and their role in the cellular interaction, remain elusive. Using our new method of microfluidics-based super-resolution fluorescence imaging platform we can now characterize the correlation between both nanoscale ligand-receptor interactions and tethering/rolling of cells under external shear stress. We found that cell rolling on E-selectin caused significant reorganization of the nanoscale clustering behavior of CD44 and CD43, from a patchy clusters of ~ 200 nm in size to an elongated network-like structures where for PSGL-1 the clustering size did not change significantly as it was 85 nm and after cell rolling the PSGL-1 aggregated to one side or even exhibited an increase in the footprint. Furthermore, I have established the use of 3D SR images that indicated that the patchy clusters of CD44 localize to protruding structures of the cell surface. On the other hand, a significant amount of the network-like elongated CD44 clusters observed after the rolling were located in the close proximity to the E-selectin surface. The effect of the nanoscale reorganization of the clusters on the HSPC rolling over selectins is still an open question at this stage. Nevertheless, my results further demonstrate that this mechanical force-induced reorganisation is accompanied by a large structural reorganisation of actin cytoskeleton. Our microfluidics-based SR imaging also demonstrate an essential role of the nanoscale clustering of CD44 on stable rolling behaviours of cells. Our new experimental platform enhances understanding of the relationship between nanoscopic ligand-receptor interactions and macroscopic cellular interactions, providing a foundation for characterizing complicated HSPC homingen
dc.language.isoenen
dc.subjectsuper-resolutionen
dc.subjectMicrofluidicsen
dc.subjectcell-rollingen
dc.subjectclusteringen
dc.subjectE-Selectinen
dc.subjectdSTORMen
dc.titleSuper-resolution fluorescence imaging of membrane nanoscale architectures of hematopoietic stem cell homing and migration moleculesen
dc.typeDissertationen
dc.contributor.departmentBiological and Environmental Sciences and Engineering (BESE) Divisionen
thesis.degree.grantorKing Abdullah University of Science and Technologyen
dc.contributor.committeememberFabrizio, Enzo Dien
dc.contributor.committeememberMerzaban, Jasmeenen
dc.contributor.committeememberDedecker, Peteren
thesis.degree.disciplineBioscienceen
thesis.degree.nameDoctor of Philosophyen
dc.person.id113538en
All Items in KAUST are protected by copyright, with all rights reserved, unless otherwise indicated.