Hydrodynamic trapping measures the interaction between membrane-associated molecules

Junghans, Victoria; Hladílková, Jana; Santos, Ana Mafalda; Lund, Mikael; Davis, Simon J.; Jönsson, Peter

doi:10.1038/s41598-018-30285-0

Cited by 11 publications

(19 citation statements)

References 29 publications

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“…However, it should be noted that the concentration of both CD43 and CD45 in the cell's glycocalyx is high, ~1,000 molecules/μm 2 each ( 8 , 63 ), and so interactions with neighboring proteins will cause CD45 to adopt a more upright position on the cell surface (Figure 2A ). Indeed, using Monte Carlo simulations, similar to those described in Junghans et al ( 62 ), we found that this effect would increase the effective height of CD45 by ~5 nm. Thus, the effective height depends not only on the total length of the protein, but also on other properties including flexibility and protein density.…”

Section: Hydrodynamic Trappingsupporting

confidence: 79%

“…The accumulation will also be affected by intermolecular interactions among the proteins. By relating the amount of accumulation to the trap strength at different positions it is possible to determine both the orientation as well as the intermolecular interactions from a single measurement ( 62 ).…”

Section: Hydrodynamic Trappingmentioning

confidence: 99%

“…Using hydrodynamic trapping we found that the largest CD45 isoform, membrane-anchored CD45RABC, interacts with other CD45RABC molecules as if the protein was a 40 nm long rod that is free to rotate around its anchoring point ( 62 ). This means that the average height of the protein on the surface would be ~25 nm at low protein densities (Figure 2A ).…”

Section: Hydrodynamic Trappingmentioning

confidence: 99%

“…It should be mentioned that only the interaction between the extracellular region of the proteins was studied and that the transmembrane domain of the proteins was replaced with a histidine tag that binds to nickel-chelating lipids in the SLB ( 62 ). Whereas this affects the mobility of the proteins compared to their native state on the cell surface, it should only moderately influence the steady state distribution in the hydrodynamic trap.…”

Section: Hydrodynamic Trappingmentioning

confidence: 99%

“…This topic has recently been covered in detail ( 23 ), however, it should be noted that the more repulsive the interaction between the proteins, the more pronounced the effect of crowding will be. Measuring these interactions can be done using hydrodynamic trapping ( 62 ), which will also indicate under what conditions the membrane-anchored molecules will cluster.…”

Section: Hydrodynamic Trappingmentioning

confidence: 99%

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Dimensions and Interactions of Large T-Cell Surface Proteins

et al. 2018

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The first step of the adaptive immune response involves the interaction of T cells that express T-cell receptors (TCRs) with peptide-loaded major histocompatibility complexes expressed by antigen-presenting cells (APCs). Exactly how this leads to activation of the TCR and to downstream signaling is uncertain, however. Recent findings suggest that one of the key events is the exclusion of the large receptor-type tyrosine phosphatase CD45, from close contacts formed at sites of T-cell/APC interaction. If this is true, a full understanding of how close contact formation leads to signaling would require insights into the structures of, and interactions between, large membrane proteins like CD45 and other proteins forming the glycocalyx, such as CD43. Structural insights into the overall dimensions of these proteins using crystallographic methods are hard to obtain, and their conformations on the cell surface are also unknown. Several imaging-based optical microscopy techniques have however been developed for analyzing protein dimensions and orientation on model cell surfaces with nanometer precision. Here we review some of these methods with a focus on the use of hydrodynamic trapping, which relies on liquid flow from a micropipette to move and trap membrane-associated fluorescently labeled molecules. Important insights that have been obtained include (i) how protein flexibility and coverage might affect the effective heights of these molecules, (ii) the height of proteins on the membrane as a key parameter determining how they will distribute in cell-cell contacts, and (iii) how repulsive interactions between the extracellular parts of the proteins influences protein aggregation and distribution.

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Section: Hydrodynamic Trappingsupporting

confidence: 79%

Section: Hydrodynamic Trappingmentioning

confidence: 99%

Section: Hydrodynamic Trappingmentioning

confidence: 99%

Section: Hydrodynamic Trappingmentioning

confidence: 99%

Section: Hydrodynamic Trappingmentioning

confidence: 99%

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Dimensions and Interactions of Large T-Cell Surface Proteins

et al. 2018

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The density of anionic lipids modulates the adsorption of α-Synuclein onto lipid membranes

Andersson,

Linse,

Sparr

et al. 2024

Biophysical Chemistry

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Single-cell measurements of two-dimensional binding affinity across cell contacts

Chouliara

Junghans

Dam

et al. 2021

Biophysical Journal

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The two-dimensional (2D) affinity between protein molecules across contacting cells is a key parameter regulating and initiating several cellular processes. However, measuring 2D affinity can be challenging, and experimental data are limited. In addition, the obtained 2D affinities are typically averaged over the cell population. We here present a method to measure 2D affinity on single cells binding to polyhistidine-tagged fluorescent ligands anchored to a supported lipid bilayer (SLB). By decreasing the density of ligands in the SLB using imidazole, a new steady-state accumulation in the contact is obtained, and from this change, both the 2D affinity and the number of receptors on the cell can be determined. The method was validated on an SLB containing rat CD2 binding to the rat CD48 mutant T92A expressed on Jurkat T cells. The addition of imidazole did not influence the average 2D affinity (1/K d ), and the spread in affinities within the cell population was low, K d ¼ 4.9 5 0.9 molecules/ mm 2 (mean 5 SD), despite an order of magnitude spread in ligand accumulation because of differences in receptor density. It was also found that cell contact size increased both with ligand density and with the number of receptors per cell but that the contact size stayed approximately constant when lowering the ligand density, above a density of around 10 rat CD2 molecules/mm 2 , after the contact first had formed, indicative of a heterogeneous process. In summary, this method not only allows for single-cell affinities to be measured, but it can also reduce measurement and analysis time and improve measurement accuracy. Because of the low spread in 2D K d within the cell population, the analysis can further be restricted to the cells showing the strongest binding, paving the way for using this method to study weak binding events.

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Hydrodynamic trapping measures the interaction between membrane-associated molecules

Cited by 11 publications

References 29 publications

Dimensions and Interactions of Large T-Cell Surface Proteins

Dimensions and Interactions of Large T-Cell Surface Proteins

The density of anionic lipids modulates the adsorption of α-Synuclein onto lipid membranes

Single-cell measurements of two-dimensional binding affinity across cell contacts

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