Introduction: Session Description: As a Society, we cannot escape the identity crisis we have confronted in the past - what is pathology and how do pathologists fit into the basic framework of biomedical science? This is an ongoing challenge that requires our members to educate others regarding the nature of the discipline of experimental pathology and how our research describes and investigates the pathology, pathogenesis, and pathophysiology of specific diseases at the molecular, cellular, organ, and organismal level. Overcoming this identity crisis requires effort on the part of each ASIP member and our success will be evident as we continue to attract bright and enthusiastic young investigators into the diverse field of experimental pathobiology.
The American Society for Investigative Pathology presents I Am An ASIP Member and This Is My Science a dynamic and inspiring session featuring ASIP Scientists on the Cutting Edge of Discovery briefly, present their research, accomplishments, career journeys, and service to ASIP. This session highlights the diversity among our membership, and provides trainees, young scientists, pathologists, and the members of the larger scientific community the opportunity to become inspired by Trailblazers in the field of investigative pathology.
Kristen Engevik (Baylor College of Medicine), Jessica Digrazia (Medical University of South Carolina), Izumi Kaji (Vanderbilt University Medical Center), Amy Engevik (Medical University of South Carolina)
Background: Intestinal enterocytes have a complex apical membrane consisting of actin rich protrusions known as microvilli. Microvilli increase the surface area of the intestine and facilitate absorption of nutrients and water. Enterocytes have approximately 1000 microvilli lining their apical membrane which is collectively known as the brush border. To accommodate a multitude of microvilli on a single cell, the microvilli must be densely packed and highly organized. The brush border assembly is tightly regulated by the intermicrovillar adhesion complex (IMAC). IMAC forms physical adhesion links at the tips of microvilli, connecting the distal tips of adjacent microvilli to control packing and microvillar length. IMAC is composed of protocadherins, such as protocadherin 24 (CDHR2) and mucin like protocadherin (CDHR5), and scaffolding proteins USH1C, ANKS4B and Myosin 7b (Myo7b). Defects in IMAC complexes results in stunted microvilli and has been linked in the ileum to Crohn’s disease (a subset of inflammatory bowel disease). Myosin 5b (Myo5b) is a molecular motor that is critical for the delivery of diverse cargo to the apical membrane of enterocytes. We hypothesized that loss of Myo5b in vivo would result in alterations in the localization of key proteins involved in the IMAC.
Methods: We used germline Myo5b KO mice, littermate controls and adult intestinal specific tamoxifen inducible VillinCreERT2;Myo5bflox/flox (Myo5b KO) mice and Myo5bflox/flox (control mice) to determine the localization of IMAC proteins following loss of Myo5b in the small intestine.
Results: RNA sequencing revealed decreased expression of CDHR2, CDHR5, USH1C and ANKS4B in both germline and adult inducible Myo5b KO mice compared to control mice. Myo7b mRNA expression was unchanged between Myo5b KO mice and control mice. Immunostaining for CDHR2 in neonatal germline Myo5b KO mice showed loss of apical CDHR2 in the brush border compared to littermate controls. CDHR5 localized to the brush border in neonatal control mice, but in neonatal germline Myo5b KO mice CDHR5 was detected below the apical membrane in the subapical compartment. In neonatal control mice, USH1C was observed to localize to the tips of microvilli as identified by gamma actin staining to delineate the actin rich brush border. USH1C was observed on the apical membrane of germline Myo5b KO mice however, USH1C did not appear to be enriched at the tips of microvilli. ANKS4B had decreased apical expression in germline Myo5b KO mice compared to littermate control mice. Similarly, there was a decrease in Myo7b staining on the apical membrane of germline Myo5b KO mice and adult inducible Myo5b KO mice compared to control mice.
Conclusions: These data indicate that Myo5b plays a key role in proper brush border formation and packing of microvilli in vivo and suggests that mutations in Myo5b likely impact intestinal barrier function.
Loss of Myo5b results in a decrease in intermicrovillar adhesion complex components at the apical membrane in germline Myo5b KO mice. CDHR2 (A), USHC1C (B), CDHR5 (C), ANKS4B (D), P-ERM and gamma actin staining showed these proteins correctly localized in control mice. Myo5b KO mice CDHR2, USH1C, CDHR5 and ANKS4B had decreased apical localization and USH1C was not enriched at the tips of microvilli. Graphs show RNA sequencing results in control and Myo5b KO mice. n=4-6 mice per group, *p < 0.05
