Figure 7: Long-term timelapse imaging reveals that microvilli accumulation at cell margins leads accumulation in medial regions during differentiation.

(A) MaxIP spinning disk confocal stills of live mCherry-ESPN expressing CL4 cells at t = 0, 24, and 43 hrs from a 43-hour acquisition. Intensity coded images show Fire LUT intensity profile of the mCherry-ESPN channel. Intensity scales from low (0; dark purple) to high (255; yellow/white) as denoted by LUT profile. Zooms at each time point are outlined by dashed boxes, with marginal and medial zones as marked. (B) Paired mean marginal and medial mCherry-ESPN intensities (16-bit gray value) at 0 and 24 hrs from three independent movies from n = 11 cells (movie 1), n = 8 cells (movie 2), and n = 11 cells (movie 3). The cut axes accounts for background mCherry signal. Mean Δ for the marginal and medial zones are denoted on each graph. (C) Mean ESPN intensity change over 24 hrs for medial (22.5 ± 5.7) and marginal (42.6 ± 3.5) zones. Error bars represent mean ± SEM. * p = 0.275 unpaired t-test. Scale bars: 20 μm (A-C), 10 μm (zooms). (D) An adhesion-based model for the marginal stabilization of microvilli during brush border assembly. Microvilli on nascent transporting epithelial cells initially organize into medial and marginal populations. Medial microvilli are highly motile while marginal microvilli are stable and stand at an orientation more vertical to the apical surface. Transjunctional CDHR2/CDHR5 heterophilic adhesion complexes span cell junctions and link marginal microvilli of neighboring cells. These complexes are long-lived, which leads to the accumulation of microvilli at the edges of cells that, in turn, may result in outside-in packing during differentiation.