Force-activated adherence, (the increase in cell-to-cell or cell-to-substrate binding after application of physical force) is common in many biomedical settings, including pathogenic and environmental biofilms, thrombogenesis, and mammalian cell adhesion. It is also a desirable property for nanofabrication of materials and flow-sensitive devices. However, we have little understanding of the molecular structures that underlie the phenomenon. We recently discovered functional amyloid-forming sequences in Candida albicans Als5p and other yeast cell adhesion proteins from many gene families. These amyloid sequences are required for formation of strong cell-to-cell adhesive bonds, and have an unusual composition being rich in Ile, Val, and Thr. The adhesins are force-activated, and the amyloid sequences mediate formation of "nanodomains" of adhesin molecules on the cell surface. Bioinformatics studies demonstrate similar sequences to be widespread among eukaryote cell adhesion molecules. Therefore, our objective in this proposal is to understand the molecular roles of amyloid sequences in force activation of fungal cell adhesins. Our central hypothesis is that these novel amyloid sequences cause force-sensitive clustering of adhesion molecules to form cell surface regions conferring strong adhesive interactions between cells. We have assembled the tools to carry out aims that will test 3 working hypotheses: 1) that Ile, Val, Thr-rich sequences are specific for force-dependent activation. We will assay the effects of substitutions of other amyloid-forming sequences on protein stability and activation. 2) That the sequence of Ile, Val, Thr-rich amyloids is less important than amino acid composition in the activity of the adhesins. Sequence variants will be tested in adhesion assays. 3) That the T domain of Als proteins acts as a force-sensitive folding switch, which unfolds to expose the amyloid sequence under extension force. The amyloid sequence in Als adhesins is in the highly conserved T domain. The wild- type sequence and amyloid-disrupted V326N mutant T domain will be embedded in other surface protein, including a GFP reporter and an adhesin constructed from mammalian mannose-specific lectins. Successful completion of this work will lead to basic understanding of the newly-discovered role of amyloids in force-responsive cell adhesion phenomena.
Specific Aim 2 will generate a search criterion for discrimination between potentially functional force-sensitive amyloid assembly systems, and fortuitous sequences.
Specific aim 3 will produce model systems for assay of role of amyloids in cell adhesion proteins in general. Knowledge generated in Aims 1 and 3 will lead to strategies for intervention in biofilm formation or other conditions in which it is desirable to prevent robust adhesion (desirable in treatment of infectious diseases or in metastasis). Conversely, the work will provide a new structural module and capability for regulating cell interactions in nanofabrication and tissue engineering.

Public Health Relevance

We have recently discovered that amyloid formation can make cell adhesion more effective by creating high avidity adhesion nanodomains on the cell surface. We propose to determine the specific sequences that promote nanodomain creation and maintenance. This knowledge will help us to control cell adhesion in biofilms, microbial infections, immune response, and nanofabrication.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
3R01GM098616-03S1
Application #
8841945
Study Section
Program Officer
Nie, Zhongzhen
Project Start
2012-06-01
Project End
2016-03-31
Budget Start
2014-04-01
Budget End
2015-03-31
Support Year
3
Fiscal Year
2014
Total Cost
$48,912
Indirect Cost
$17,758
Name
Brooklyn College
Department
Biology
Type
Schools of Arts and Sciences
DUNS #
620127691
City
New York
State
NY
Country
United States
Zip Code
11210
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Garcia-Sherman, Melissa C; Lysak, Nataliya; Filonenko, Alexandra et al. (2014) Peptide detection of fungal functional amyloids in infected tissue. PLoS One 9:e86067
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