Figure 2.

(A) Distribution of structural proteins during rBM-induced acinar morphogenesis. Confocal fluorescence images (0.2-μm optical sections) of collagen IV, β-catenin, and Ki-67 in HMECs embedded within a rBM for 3–4 days (proliferating cells; a–c), and for 7–10 days (growth-arrested acini; d–f). Coincident with growth arrest and acinar morphogenesis, HMECs deposited an organized endogenous collagen IV-rich BM (a vs. d), whereas β-catenin relocalized from the cytosol and basal plasma membrane to sites of cell–cell adhesion (b vs. e). Acinar morphogenesis was associated with cell cycle exit, as indicated by the loss of Ki-67 staining (c vs. f). (B). Spatial analysis of NuMA and splicing factor SRm160 redistribution during rBM-induced acinar morphogenesis. Confocal Texas red fluorescence images (0.2-μm optical sections) of NuMA (a–c) and double-labeled NuMA (Texas red), and fluorescein isothiocyanate (FITC) green-stained SRm160 (a′, a′′, b′, b′′, c′, and c′′) in HMT-3522 cells proliferating (a, a′, and a′′) and undergoing morphogenesis (b, b′, b′′, c, c′, and c′′) in response to a rBM. In proliferating cells, NuMA was diffusely distributed (a) and did not colocalize with SRm160 (a′ and a′′). After growth arrest, NuMA coalesced into foci of increasing size (0.2–2 μm; f) in association with the establishment of mature tissue-like structures (acini; b and c) Nine nuclei are shown in b. Only the larger NuMA foci observed in late morphogenesis fully colocalized with SRm160 (b′, b′′, c′, and c′′). (d) In the ductal and acinar HMECs of the mammary gland, in vivo, NuMA was localized in foci with a size distribution comparable to that observed in most of the HMEC nuclei of differentiating rBM cultures shown in b. (e) Western blot analysis of NuMA and Lamin B showed no difference in protein expression or size between proliferating and growth-arrested HMECs grown within rBMs. Arrows indicate nuclei. (Bars = 10 μm.)