Determine How Contractility Enables Rapid Stem Cell Responses in the Colon.

Focal adhesions have been shown as essential for stem cell commitment during development and gut homeostasis. Our initial findings provide intriguing insights into the distribution of adhesion-associated actomyosin contractility and how it relates to differences in signaling profiles inside and outside the stem cell compartment in the human colon. Specifically, we have observed a notable shift in the contractility of cells, observing a change from more apical contractility in the stem cell compartment to a focal nature in the differentiated compartment. In the murine small intestine, a distinct change occurs in cell contractility as cells move from the base of the crypt upwards: the level of total myosin IIA, a protein involved in contractility, doubles, while, at the same time, the amount of apical myosin IIA decreases by an equivalent degree. In our patient-derived colonoids, we observe this same change in phenotype as cells move up and out of the stem cell compartment, and this shift in contractility is closely linked to the relocation of the actin polymerizing protein Ena/VASP-like (EVL), which is abundant at the apical membrane of the stem cells but absent at focal adhesions. Ena/VASP proteins have been shown to regulate contractility at both cell-cell junctions and focal adhesions. This relocation of contractility proteins is not just a physical change but also coincides with a significant alteration in cellular signaling. We have noticed a transition from Akt signaling, which is commonly associated with stem cell renewal, to Erk signaling, which is often linked to cell differentiation and survival. This switch from Akt to Erk signaling during stem cell commitment in the gut has also been observed in murine intestinal tissues, but the mechanisms controlling of this change have yet to be established.

Erk signaling has been established as both an inducer of and a responder to changes in cell-substrate interactions. We propose that the change in mechanical profiles in the cells of the epithelium drives signaling profiles that regulate the proliferation and differentiation of stem cells. Kinases which regulate focal adhesion turnover and maturation, such as focal adhesion kinase (FAK), have also been shown to drive cell fate decisions in the gut epithelium. These mechanical signals seem to drive cell fate decisions, particularly during the crucial phase of stem cell commitment. By influencing the dynamic interplay between Erk and Akt signaling pathways, these mechanical signals appear to dictate whether a stem cell will self-renew and continue to proliferate or start differentiating.

This project is designed to unravel the interplay of mechanical signals and intracellular signaling pathways. We aim to uncover how the balance between Erk and Akt signaling is altered during the critical phase of stem cell development and the implications of this balance for stem cell fate. Understanding these mechanisms is vital, as it could provide profound insights into how stem cells differentiate and how this process can be influenced or controlled, which has far-reaching implications for regenerative medicine and cellular therapy.