Related Experiment Video
Updated: Aug 1, 2026
07:53
Actin Co-Sedimentation Assay; for the Analysis of Protein Binding to F-Actin
Published on: March 28, 2008
The actin cytoskeleton regulates LFA-1 ligand binding through avidity rather than affinity changes.
Y van Kooyk1, S J van Vliet, C G Figdor
1Department of Tumor Immunology, University Hospital Nijmegen St. Radboud, 6525 EX Nijmegen, The Netherlands. Y.vanKooyk@dent.kun.nl
The Journal of Biological Chemistry
|September 10, 1999
Summary
Cytoskeleton regulation of lymphocyte function-associated antigen-1 (LFA-1) adhesion is primarily driven by receptor clustering, not altered ligand binding affinity. This finding impacts understanding of leukocyte adhesion and immune responses.
Area of Science:
- Immunology
- Cell Biology
- Biochemistry
Background:
- Leukocyte adhesion is crucial for immune responses.
- Lymphocyte function-associated antigen-1 (LFA-1) is a key adhesion receptor.
- Cytoskeletal regulation of LFA-1 avidity and affinity is not fully understood.
Purpose of the Study:
- To investigate the role of the cytoskeleton in regulating LFA-1 avidity and affinity.
- To determine if LFA-1-mediated adhesion is regulated by receptor diffusion/clustering or altered ligand binding affinity.
Main Methods:
- Generated mutant cytoplasmic LFA-1 receptors.
- Expressed mutants in K562 cells.
- Assessed ICAM-1-mediated adhesion, receptor distribution, and ligand-binding affinity.
Main Results:
- Cytoplasmic tail deletion mutants of LFA-1 were constitutively active.
- Mutants exhibited clustered cell surface distribution but unaltered ligand-binding affinity.
- Disrupting the cytoskeleton did not affect mutant binding affinity or adhesion.
Conclusions:
- LFA-1-mediated cell adhesion to ICAM-1 is predominantly regulated by receptor clustering.
- Affinity alterations do not necessarily correlate with strong ICAM-1 binding.
- LFA-1 coupling to the actin cytoskeleton is not required for ICAM-1 binding in these mutants.
Related Concept Videos
02:40Ligand Binding Sites
Proteins are dynamic macromolecules that carry out a wide variety of essential processes; however, the activities of most proteins depend on their interactions with other molecules or ions, known as ligands.
Protein-ligand interactions are quite specific; even though numerous potential ligands surround a cellular protein at any given time, only a particular ligand can bind to that protein. Moreover, a ligand binds only to a dedicated area on the surface of the protein, known as the...
Protein-ligand interactions are quite specific; even though numerous potential ligands surround a cellular protein at any given time, only a particular ligand can bind to that protein. Moreover, a ligand binds only to a dedicated area on the surface of the protein, known as the...
01:26Introduction to Actin
Actin is a highly conserved cytoskeletal protein found abundantly in eukaryotic cells. It constitutes 10% weight of the total cellular protein in muscle cells, while in non-muscle cells, it is lower and makes up around 1–5 percent of the total cell protein. Actin found in the unicellular amoebae and complex multicellular animals is around 80% similar, demonstrating their conservation over a billion years of evolution. Actin coding genes are conserved within species and across different species.
01:19Actin Filament Depolymerization
Actin filaments (F-actin) are composed of actin subunits. The dissociation of actin monomers can occur from either end of F-actin. The rate of dissociation is faster from the minus-end or the pointed end, where the actin subunits exist with a bound ADP, together known as ADP-actin. The depolymerization of F-actin is aided by proteins, including the actin-depolymerizing factor (ADF) and cofilin family of proteins, gelsolin, and glia maturation factor (GMF).
In F-actin, the ADF/cofilin proteins...
In F-actin, the ADF/cofilin proteins...
01:11Formation of Higher-order Actin Filaments
The polymerization of G-actin monomers into filamentous F-actin is a multi-step process. Once the F-actins are formed, they can bundle together in different arrangements to form higher-order networks and regulate cellular functions. Common examples include the formation of lamellipodia and filopodia at the cell's leading edge by actin reorganization in a migrating cell. The microvilli on the brush border epithelial cells are also formed through the F-actin network.
The high-order actin networks...
The high-order actin networks...
01:39Mechanism of Filopodia Formation
Filopodia are thin, actin-rich cellular protrusions that play an important role in many fundamental cellular functions. They vary in their occurrence, length, and positioning in different cell types, suggesting their diverse roles.
Their main function is to guide migrating cells during normal tissue morphogenesis or cancer metastasis by recognizing and making initial contacts with the extracellular matrix. However, they can also act as stationary cell anchors or help to establish communication...
Their main function is to guide migrating cells during normal tissue morphogenesis or cancer metastasis by recognizing and making initial contacts with the extracellular matrix. However, they can also act as stationary cell anchors or help to establish communication...
01:17Intracellular Signaling Affects Focal Adhesions
Integrins act both as extracellular input receivers and as intracellular processing activators. As their name suggests, integrins are entirely integrated into the membrane structure. Their hydrophobic membrane-spanning regions interact with the phospholipid bilayer's hydrophobic region. These membrane receptors provide extracellular attachment sites for effectors like hormones and growth factors. They activate intracellular response cascades when their effectors are bound and active.
Some...
Some...