@article {Chanez-Paredes2022.10.13.512159,
	title = {Selective perijunctional MLCK1 recruitment in Crohn{\textquoteright}s disease: Identification of essential structural domains},
	journal = {bioRxiv},
	year = {2022},
	publisher = {Cold Spring Harbor Laboratory},
	abstract = {Intestinal epithelia express two long MLCK splice variants, MLCK1 and MLCK2. We have previously shown that disruption of inflammation-induced MLCK1 recruitment to the perijunctional actomyosin ring prevents barrier loss and attenuates disease progression. Here we sought to define the domains responsible for distinct MLCK1 and MLCK2 behaviors. Quantitative analysis of human biopsies demonstrated specific increases in MLCK1 expression and perijunctional localization in Crohn{\textquoteright}s disease. When expressed in cultured intestinal epithelial cells, we found, as expected, that MLCK1 is most concentrated at the perijunctional actomyosin ring. In contrast, MLCK2 is predominantly associated with basal F-actin stress fibers. Immunoglobulin-cell adhesion molecule domain 3 (IgCAM3) must be critical for MLCK1 recruitment, as that domain is incomplete in MLCK2. Consistent with this, truncation mutants consisting of N-terminal IgCAM domains 1-4, without C-terminal catalytic domains, localized similarly to full-length MLCK1 and MLCK2, respectively. Further mutagenesis allowed identification of IgCAM2 and IgCAM3 domains as the minimal region required for MLCK1 recruitment. Although IgCAM3 does not concentrate perijunctionally, it can act as a dominant negative effector that limits steady-state and TNF-induced MLCK1 recruitment and barrier loss. Together, the demonstration of selective MLCK1 upregulation and perijunctional recruitment in Crohn{\textquoteright}s disease and identification of domains required for perijunctional MLCK1 recruitment provide a conceptual understanding and structural data needed for development of therapeutic means of blocking MLCK1-mediated barrier loss without the toxicity of enzymatic MLCK inhibition.SIGNIFICANCE STATEMENT Recent work has demonstrated that long myosin light chain kinase isoform 1 (MLCK1) recruitment to the perijunctional actomyosin ring is a critical component of inflammation-induced intestinal barrier loss. Chanez-Paredes et al. show that this occurs in Crohn{\textquoteright}s disease and define the essential structural elements that direct MLCK1 recruitment, thereby creating a foundation for therapeutic interruption of MLCK1 recruitment in disease.Competing Interest StatementJRT is a founder of Thelium Therapeutics and a consultant for Entrinsic and Kallyope.},
	doi = {10.1101/2022.10.13.512159},
	url = {https://www.biorxiv.org/content/early/2022/10/14/2022.10.13.512159},
	author = {Chanez-Paredes, Sandra D. and Abtahi, Shabnam and Zha, Juanmin and Zuo, Li and He, Weiqi and Turner, Jerrold R.}
}
