The Role of Cell Deformation During Selectin-Mediated Neutrophil Rolling

Selectin-mediated neutrophil rolling on endothelial cells is an important step in the biological processes of inflammation and thrombosis. Conversely, it has also been implicated in the pathogenesis of various cardiovascular diseases. While there are many factors that affect this phenomenon, shear stress is among the most significant. Previous studies have demonstrated the effects of shear stress on the selectin/ligand interactions that sustain neutrophil rolling. However, the effects of cell deformation on neutrophil rolling are still under investigation. In this study, the effects of cell deformation on selectin-mediated rolling are investigated on two scales: (I)deformation on a local scale such as contact zone deformation or membrane tether formation and (II)deformation of the cell body on a global scale.We present reflective interference contrast microscopy (RICM) as a low-cost alternative to current microscopy techniques used to visualize cell footprints during rolling (i.e. quantitative dynamic footprinting). RICM visualized neutrophil contact areas similar to those reported by qDF and also visualized the formation of membrane tethers from distinct adhesion points. We also use RICM to show that membrane tether formation and neutrophil contact area are dependent on shear stress rather than shear rate.Alternatively, using differential interference contrast (DIC) microscopy we demonstrate that increased membrane tether lifetime correlates with increasing P-selectin density while increased tether length correlates with increased shear stress. We also report the characterization of membrane tether formation on recombinant human P- and E-selectin-Fc chimeras.Cellular deformation on a global scale has been suggested to occur as a result of the shearing force applied to an adherent cell by hemodynamic flow. Previous computational studies have related this shear effect to the ratio between the diameter of the cell (Dc) and the height of the channel being perfused (H). Although this Dc/H ratio can be large for neutrophils in post-capillary venules where leukocyte rolling typically occurs, many in vitro studies have used flow chambers where Dc/H is small (< 0.1).To investigate leukocyte rolling in chambers with dimensions similar to post-capillary venules, we fabricated microfluidic chambers with various cross-sectional dimensions that required perfusion rates as low as 40 nL/min. We observed a significant decrease in neutrophil rolling velocity on P-selectin that correlated with decreasing chamber height (increasing Dc/H). Comparable studies with fixed cells and PSGL-1 coated microspheres, as well as contact area measurements made with RICM, demonstrated this velocity decrease was related to cell deformability.Likewise, we fabricated bifurcated microfluidic chambers to investigate the impact of chamber geometry on neutrophil adhesion. Neutrophils accumulated at the bifurcation apex in P-selectin coated chambers at shear stresses as high as 100 dynes/cm2. Similarly, we used fixed cells to demonstrate that this accumulation is related to cellular deformation. We also report that adherent cell accumulation is dependent on specific molecular interactions, P-selectin coating concentration and the geometric properties of the chambers (e.g. bifurcation angle and channel cross-section dimensions)

Near-infrared emitting quantum dots for cellular and vascular fluorescent labeling in in vivo multiplexed imaging studies

Thesis (Ph. D.)--Harvard-MIT Division of Health Sciences and Technology, 2011.Cataloged from PDF version of thesis.Includes bibliographical references (p. 199-217).In vivo multimodal, multiplexed microscopy allows real-time observation of hematopoietic cells, their stem and progenitor cells and metastatic cancer cells in their native bone marrow (BM) environment. Multiplexing has made possible detailed studies of the BM's microarchitecture, which helps define the niche of these cells; it has nonetheless been limited by the paucity of suitable probes fluorescent in the near-infrared spectrum that is favored by tissue optics. This project attempts to address this problem by developing cellular and vascular fluorescent imaging probes comprised of semiconductor nanocrystals, or quantum dots (QDs), with tunable fluorescence between 65o-8oonm and exhibiting photostability, robust quantum yield and narrow fluorescence profiles that are critical for such applications. The synthesis of alloyed CdTexSe1 x QDs will be detailed in the thesis. Reproducibility and workability in subsequent steps are emphasized in the methods. Special attention is also paid to the difference between working with alloyed versus single semiconductor QDs, especially the need to achieve physical and spectral uniformity when composition and its gradient are also variable. The steps for creating biological probes from these QD fluorophores are also described. They include overcoating, water solubilization and functionalization for cellular uptake and vascular retention. Finally, the thesis returns to its motivation and reports novel methods, developed using NIR QD vascular imaging probes, for visualizing in vivo 3-D imaging data of the murine BM and characterizing the tissue's architecture. Measuring the Euclidean distance between BM osteoblasts and blood vessels is presented to exemplify a potential platform for describing the geographic relationships between cells, molecules and structural components in any tissue.by Juwell Wendy Wu.Ph.D

Technology 2001: The Second National Technology Transfer Conference and Exposition, volume 2

Proceedings of the workshop are presented. The mission of the conference was to transfer advanced technologies developed by the Federal government, its contractors, and other high-tech organizations to U.S. industries for their use in developing new or improved products and processes. Volume two presents papers on the following topics: materials science, robotics, test and measurement, advanced manufacturing, artificial intelligence, biotechnology, electronics, and software engineering

Microfluidic Reduction of Osmotic Stress in Oocyte and Zygote Vitrification.

Microfluidic cryoprotectant exchange enables vitrification of murine zygotes with superior morphology as indicated by a smoother cell surface and higher developmental competence compared to conventional methods. Bovine oocyte vitrification also benefit as evidenced by higher lipid retention. Experimental observations and mathematical analysis demonstrate that the microfluidic advantage arise predominantly from eliminating high shrinkage rates associated with abrupt and uneven exposure to vitrification solutions that readily occur in current manual protocols. The microfluidic cryoprotectant exchange method described has immediate applications for improving animal and human oocyte, zygote, and embryo cryopreservation. On a fundamental level, the clear demonstration that at the same minimum cell volume, cell shrinkage rate affects sub-lethal damage should be broadly useful for cryobiology.PhDBiomedical EngineeringUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/107056/1/davlai_1.pd

Psr1p interacts with SUN/sad1p and EB1/mal3p to establish the bipolar spindle

Regular Abstracts - Sunday Poster Presentations: no. 382During mitosis, interpolar microtubules from two spindle pole bodies (SPBs) interdigitate to create an antiparallel microtubule array for accommodating numerous regulatory proteins. Among these proteins, the kinesin-5 cut7p/Eg5 is the key player responsible for sliding apart antiparallel microtubules and thus helps in establishing the bipolar spindle. At the onset of mitosis, two SPBs are adjacent to one another with most microtubules running nearly parallel toward the nuclear envelope, creating an unfavorable microtubule configuration for the kinesin-5 kinesins. Therefore, how the cell organizes the antiparallel microtubule array in the first place at mitotic onset remains enigmatic. Here, we show that a novel protein psrp1p localizes to the SPB and plays a key role in organizing the antiparallel microtubule array. The absence of psr1+ leads to a transient monopolar spindle and massive chromosome loss. Further functional characterization demonstrates that psr1p is recruited to the SPB through interaction with the conserved SUN protein sad1p and that psr1p physically interacts with the conserved microtubule plus tip protein mal3p/EB1. These results suggest a model that psr1p serves as a linking protein between sad1p/SUN and mal3p/EB1 to allow microtubule plus ends to be coupled to the SPBs for organization of an antiparallel microtubule array. Thus, we conclude that psr1p is involved in organizing the antiparallel microtubule array in the first place at mitosis onset by interaction with SUN/sad1p and EB1/mal3p, thereby establishing the bipolar spindle.postprin

Removal of antagonistic spindle forces can rescue metaphase spindle length and reduce chromosome segregation defects

Regular Abstracts - Tuesday Poster Presentations: no. 1925Metaphase describes a phase of mitosis where chromosomes are attached and oriented on the bipolar spindle for subsequent segregation at anaphase. In diverse cell types, the metaphase spindle is maintained at a relatively constant length. Metaphase spindle length is proposed to be regulated by a balance of pushing and pulling forces generated by distinct sets of spindle microtubules and their interactions with motors and microtubule-associated proteins (MAPs). Spindle length appears important for chromosome segregation fidelity, as cells with shorter or longer than normal metaphase spindles, generated through deletion or inhibition of individual mitotic motors or MAPs, showed chromosome segregation defects. To test the force balance model of spindle length control and its effect on chromosome segregation, we applied fast microfluidic temperature-control with live-cell imaging to monitor the effect of switching off different combinations of antagonistic forces in the fission yeast metaphase spindle. We show that spindle midzone proteins kinesin-5 cut7p and microtubule bundler ase1p contribute to outward pushing forces, and spindle kinetochore proteins kinesin-8 klp5/6p and dam1p contribute to inward pulling forces. Removing these proteins individually led to aberrant metaphase spindle length and chromosome segregation defects. Removing these proteins in antagonistic combination rescued the defective spindle length and, in some combinations, also partially rescued chromosome segregation defects. Our results stress the importance of proper chromosome-to-microtubule attachment over spindle length regulation for proper chromosome segregation.postprin