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Identification of CALML4 as a Novel Component of the Intermicrovillar Adhesion Complex that Regulates Intestinal Brush Border Assembly

Choi, Myoung Soo
http://rave.ohiolink.edu/etdc/view?acc_num=toledo1532947508319487

Abstract Details

2018, Master of Science, University of Toledo, Biology (Cell-Molecular Biology).
During their terminal differentiation program, intestinal enterocytes form an apical brush border (BB): a tightly packed array of lumen-oriented microvilli that function in nutrient absorption and host-cell defense. Assembly of a highly-ordered BB is driven by a protocadherin-based adhesion complex, known as the intermicrovillar adhesion complex (IMAC), that mediates adhesion between neighboring microvilli during BB formation. We used the innate adhesion of the IMAC protocadherins as an affinity purification step to recover endogenous IMAC, and have identified calmodulin-like protein 4 (CALML4) as a novel component of this complex. CALML4 is a small EF hand protein that has yet to be characterized in the literature. Endogenous CALML4 exhibits striking localization to the distal tip of BB microvilli, the site of IMAC function, in native intestinal tissue and CACO-2BBE cell culture enterocytes. Overexpression of EGFP-tagged CALML4 in CACO-2BBE cells recapitulates this localization. Through a series of pull-down assays, we identified that CALML4 incorporates into the IMAC by associating with the neck region of Myo7b as a myosin light chain. Refined mapping studies revealed that CALML4 associates with the third IQ motif (IQ3) in the Myo7b neck region. Interestingly, however, binding of iv CALML4 to IQ3 is dependent upon the presence of neighboring IQ motifs, suggesting that CALML4 exhibits an atypical myosin-binding mode compared to other known myosin light chains. Consistent with CALML4 functioning as light chain for Myo7b, CALML4 fails to target to the BB in Myo7b knockdown (KD) CACO-2BBE cell lines and in a Myo7b knockout (KO) mouse. Stable KD of CALML4 in CACO-2BBE cells disrupts proper BB assembly, phenocopying the loss of Myo7b itself. This suggests that CALML4 may be necessary for Myo7b in vivo function. Biophysical analysis of recombinant CALML4 revealed that it is monomeric and lacks the ability to bind calcium with high affinity. CALML4, therefore, is unlikely to be regulated by calcium directly within the IMAC. We present further evidence that CALML4 functions as a light chain for Myo7a in the homologous Usher syndrome adhesion complex (USAC) that functions in sensory epithelia. The USAC is primarily characterized in inner ear hair cells, where it is responsible for assembling arrays of specialized microvilli, known as stereocilia, into `hair bundles’ that decode sound information into neural signals. Defects in the USAC result in Usher syndrome Type 1 (USH1), a severe form of deaf-blindness. Excitingly, CALML4 is one of 27 candidate genes for USH1H, a subtype of USH1 whose genetic cause is currently unknown. In sum, our data supports the hypothesis that CALML4 functions as a myosin light chain for Myo7a and Myo7b in the USAC and IMAC, respectively. CALML4 is, therefore, the second component shared genetically between these homologous adhesion complexes.
William Scott Crawley (Committee Chair)
Song-Tao Liu (Committee Member)
Deborah Chadee (Committee Member)
Qian Chen (Committee Member)
111 p.
Biology
CALML4, calmoduline, myosin, myosin 7b, IMAC

Recommended Citations

Citations

  • Choi, M. S. (2018). Identification of CALML4 as a Novel Component of the Intermicrovillar Adhesion Complex that Regulates Intestinal Brush Border Assembly [Master's thesis, University of Toledo]. OhioLINK Electronic Theses and Dissertations Center. http://rave.ohiolink.edu/etdc/view?acc_num=toledo1532947508319487

    APA Style (7th edition)

  • Choi, Myoung Soo. Identification of CALML4 as a Novel Component of the Intermicrovillar Adhesion Complex that Regulates Intestinal Brush Border Assembly. 2018. University of Toledo, Master's thesis. OhioLINK Electronic Theses and Dissertations Center, http://rave.ohiolink.edu/etdc/view?acc_num=toledo1532947508319487.

    MLA Style (8th edition)

  • Choi, Myoung Soo. "Identification of CALML4 as a Novel Component of the Intermicrovillar Adhesion Complex that Regulates Intestinal Brush Border Assembly." Master's thesis, University of Toledo, 2018. http://rave.ohiolink.edu/etdc/view?acc_num=toledo1532947508319487

    Chicago Manual of Style (17th edition)

toledo1532947508319487
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