Interplay between the plasma membrane and cell-cell adhesion maintains epithelial identity for correct polarised cell divisions.

Polarised epithelial cell divisions represent a fundamental mechanism for tissue maintenance and morphogenesis. Morphological and mechanical changes in the plasma membrane influence the organisation and crosstalk of microtubules and actin at the cell cortex, thereby regulating the mitotic spindle machinery and chromosome segregation. Yet, the precise mechanisms linking plasma membrane remodelling to cell polarity and cortical cytoskeleton dynamics to ensure accurate execution of mitosis in mammalian epithelial cells remain poorly understood. Here, we manipulated the density of mammary epithelial cells in culture, which led to several mitotic defects. Perturbation of cell-cell adhesion formation impairs the dynamics of the plasma membrane, affecting the shape and size of mitotic cells and resulting in defects in mitotic progression and the generation of daughter cells with aberrant architecture. In these conditions, F- actin-astral microtubule crosstalk is impaired, leading to mitotic spindle misassembly and misorientation, which in turn contributes to chromosome mis-segregation. Mechanistically, we identify S100 Ca2+-binding protein A11 (S100A11) as a key membrane-associated regulator that forms a complex with E-cadherin (CDH1) and the leucine-glycine-asparagine repeat protein LGN (also known as GPSM2) to coordinate plasma membrane remodelling with E-cadherin-mediated cell adhesion and LGN-dependent mitotic spindle machinery. Thus, plasma membrane-mediated maintenance of mammalian epithelial cell identity is crucial for correct execution of polarised cell divisions, genome maintenance and safeguarding tissue integrity.

Annexin A1 is a polarity cue that directs mitotic spindle orientation during mammalian epithelial morphogenesis.

Oriented cell divisions are critical for the formation and maintenance of structured epithelia. Proper mitotic spindle orientation relies on polarised anchoring of force generators to the cell cortex by the evolutionarily conserved protein complex formed by the Gαi subunit of heterotrimeric G proteins, the Leucine-Glycine-Asparagine repeat protein (LGN) and the nuclear mitotic apparatus protein. However, the polarity cues that control cortical patterning of this ternary complex remain largely unknown in mammalian epithelia. Here we identify the membrane-associated protein Annexin A1 (ANXA1) as an interactor of LGN in mammary epithelial cells. Annexin A1 acts independently of Gαi to instruct the accumulation of LGN and nuclear mitotic apparatus protein at the lateral cortex to ensure cortical anchoring of Dynein-Dynactin and astral microtubules and thereby planar alignment of the mitotic spindle. Loss of Annexin A1 randomises mitotic spindle orientation, which in turn disrupts epithelial architecture and luminogenesis in three-dimensional cultures of primary mammary epithelial cells. Our findings establish Annexin A1 as an upstream cortical cue that regulates LGN to direct planar cell divisions during mammalian epithelial morphogenesis.

Cultivating mentorship, cooperation, and community during COVID-19 and beyond.

Over the past year, Cell Stem Cell has introduced early-career researchers impacted by the COVID-19 pandemic and subsequent closures to our readers. One year since our first introductions, we've invited several participants to reflect on their experiences and key issues. In this Story, Salah Elias talks about the importance of mentorship, support, and community, and new platforms to facilitate connections between early career researchers.

Blimp-1/PRDM1 is a critical regulator of Type III Interferon responses in mammary epithelial cells.

The transcriptional repressor Blimp-1 originally cloned as a silencer of type I interferon (IFN)-ß gene expression controls cell fate decisions in multiple tissue contexts. Conditional inactivation in the mammary gland was recently shown to disrupt epithelial cell architecture. Here we report that Blimp-1 regulates expression of viral defense, IFN signaling and MHC class I pathways, and directly targets the transcriptional activator Stat1. Blimp-1 functional loss in 3D cultures of mammary epithelial cells (MECs) results in accumulation of dsRNA and expression of type III IFN-λ. Cultures treated with IFN lambda similarly display defective lumen formation. These results demonstrate that type III IFN-λ profoundly influences the behavior of MECs and identify Blimp-1 as a critical regulator of IFN signaling cascades.

Long-lived unipotent Blimp1-positive luminal stem cells drive mammary gland organogenesis throughout adult life.

The hierarchical relationships between various stem and progenitor cell subpopulations driving mammary gland morphogenesis and homoeostasis are poorly understood. Conditional inactivation experiments previously demonstrated that expression of the zinc finger transcriptional repressor Blimp1/PRDM1 is essential for the establishment of epithelial cell polarity and functional maturation of alveolar cells. Here we exploit a Prdm1.CreERT2-LacZ reporter allele for lineage tracing experiments. Blimp1 expression marks a rare subpopulation of unipotent luminal stem cells that initially appear in the embryonic mammary gland at around E17.5 coincident with the segregation of the luminal and basal compartments. Fate mapping at multiple time points in combination with whole-mount confocal imaging revealed these long-lived unipotent luminal stem cells survive consecutive involutions and retain their identity throughout adult life. Blimp1+ luminal stem cells give rise to Blimp1- progeny that are invariably Elf5+ERα-PR-. Thus, Blimp1 expression defines a mammary stem cell subpopulation with unique functional characteristics.

Myosin 1b and F-actin are involved in the control of secretory granule biogenesis.

Hormone secretion relies on secretory granules which store hormones in endocrine cells and release them upon cell stimulation. The molecular events leading to hormone sorting and secretory granule formation at the level of the TGN are still elusive. Our proteomic analysis of purified whole secretory granules or secretory granule membranes uncovered their association with the actomyosin components myosin 1b, actin and the actin nucleation complex Arp2/3. We found that myosin 1b controls the formation of secretory granules and the associated regulated secretion in both neuroendocrine cells and chromogranin A-expressing COS7 cells used as a simplified model of induced secretion. We show that F-actin is also involved in secretory granule biogenesis and that myosin 1b cooperates with Arp2/3 to recruit F-actin to the Golgi region where secretory granules bud. These results provide the first evidence that components of the actomyosin complex promote the biogenesis of secretory granules and thereby regulate hormone sorting and secretion.

The transcriptional repressor Blimp1 is expressed in rare luminal progenitors and is essential for mammary gland development.

Mammary gland morphogenesis depends on a tight balance between cell proliferation, differentiation and apoptosis, to create a defined functional hierarchy within the epithelia. The limited availability of stem cell/progenitor markers has made it challenging to decipher lineage relationships. Here, we identify a rare subset of luminal progenitors that express the zinc finger transcriptional repressor Blimp1, and demonstrate that this subset of highly clonogenic luminal progenitors is required for mammary gland development. Conditional inactivation experiments using K14-Cre and WAPi-Cre deleter strains revealed essential functions at multiple developmental stages. Thus, Blimp1 regulates proliferation, apoptosis and alveolar cell maturation during puberty and pregnancy. Loss of Blimp1 disrupts epithelial architecture and lumen formation both in vivo and in three-dimensional (3D) primary cell cultures. Collectively, these results demonstrate that Blimp1 is required to maintain a highly proliferative luminal subset necessary for mammary gland development and homeostasis.

Huntingtin Is Required for Epithelial Polarity through RAB11A-Mediated Apical Trafficking of PAR3-aPKC.

The establishment of apical-basolateral polarity is important for both normal development and disease, for example, during tumorigenesis and metastasis. During this process, polarity complexes are targeted to the apical surface by a RAB11A-dependent mechanism. Huntingtin (HTT), the protein that is mutated in Huntington disease, acts as a scaffold for molecular motors and promotes microtubule-based dynamics. Here, we investigated the role of HTT in apical polarity during the morphogenesis of the mouse mammary epithelium. We found that the depletion of HTT from luminal cells in vivo alters mouse ductal morphogenesis and lumen formation. HTT is required for the apical localization of PAR3-aPKC during epithelial morphogenesis in virgin, pregnant, and lactating mice. We show that HTT forms a complex with PAR3, aPKC, and RAB11A and ensures the microtubule-dependent apical vesicular translocation of PAR3-aPKC through RAB11A. We thus propose that HTT regulates polarized vesicular transport, lumen formation and mammary epithelial morphogenesis.

Mutant huntingtin affects cortical progenitor cell division and development of the mouse neocortex.

A polyglutamine expansion in huntingtin (HTT) causes the specific death of adult neurons in Huntington's disease (HD). Most studies have thus focused on mutant HTT (mHTT) toxicity in adulthood, and its developmental effects have been largely overlooked. We found that mHTT caused mitotic spindle misorientation in cultured cells by altering the localization of dynein, NuMA, and the p150(Glued) subunit of dynactin to the spindle pole and cell cortex and of CLIP170 and p150(Glued) to microtubule plus-ends. mHTT also affected spindle orientation in dividing mouse cortical progenitors, altering the thickness of the developing cortex. The serine/threonine kinase Akt, which regulates HTT function, rescued the spindle misorientation caused by the mHTT, by serine 421 (S421) phosphorylation, in cultured cells and in mice. Thus, cortical development is affected in HD, and this early defect can be rescued by HTT phosphorylation at S421.

Huntingtin regulates mammary stem cell division and differentiation.

Little is known about the mechanisms of mitotic spindle orientation during mammary gland morphogenesis. Here, we report the presence of huntingtin, the protein mutated in Huntington's disease, in mouse mammary basal and luminal cells throughout mammogenesis. Keratin 5-driven depletion of huntingtin results in a decreased pool and specification of basal and luminal progenitors, and altered mammary morphogenesis. Analysis of mitosis in huntingtin-depleted basal progenitors reveals mitotic spindle misorientation. In mammary cell culture, huntingtin regulates spindle orientation in a dynein-dependent manner. Huntingtin is targeted to spindle poles through its interaction with dynein and promotes the accumulation of NUMA and LGN. Huntingtin is also essential for the cortical localization of dynein, dynactin, NUMA, and LGN by regulating their kinesin 1-dependent trafficking along astral microtubules. We thus suggest that huntingtin is a component of the pathway regulating the orientation of mammary stem cell division, with potential implications for their self-renewal and differentiation properties.

Chromogranin A induces the biogenesis of granules with calcium- and actin-dependent dynamics and exocytosis in constitutively secreting cells.

Chromogranins are a family of acidic glycoproteins that play an active role in hormone and neuropeptide secretion through their crucial role in secretory granule biogenesis in neuroendocrine cells. However, the molecular mechanisms underlying their granulogenic activity are still not fully understood. Because we previously demonstrated that the expression of the major component of secretory granules, chromogranin A (CgA), is able to induce the formation of secretory granules in nonendocrine COS-7 cells, we decided to use this model to dissect the mechanisms triggered by CgA leading to the biogenesis and trafficking of such granules. Using quantitative live cell imaging, we first show that CgA-induced organelles exhibit a Ca(2+)-dependent trafficking, in contrast to native vesicle stomatitis virus G protein-containing constitutive vesicles. To identify the proteins that confer such properties to the newly formed granules, we developed CgA-stably-expressing COS-7 cells, purified their CgA-containing granules by subcellular fractionation, and analyzed the granule proteome by liquid chromatography-tandem mass spectrometry. This analysis revealed the association of several cytosolic proteins to the granule membrane, including GTPases, cytoskeleton-based molecular motors, and other proteins with actin- and/or Ca(2+)-binding properties. Furthermore, disruption of cytoskeleton affects not only the distribution and the transport but also the Ca(2+)-evoked exocytosis of the CgA-containing granules, indicating that these granules interact with microtubules and cortical actin for the regulated release of their content. These data demonstrate for the first time that the neuroendocrine factor CgA induces the recruitment of cytoskeleton-, GTP-, and Ca(2+)-binding proteins in constitutively secreting COS-7 cells to generate vesicles endowed with typical dynamics and exocytotic properties of neuroendocrine secretory granules.

The PACAP-regulated gene selenoprotein T is highly induced in nervous, endocrine, and metabolic tissues during ontogenetic and regenerative processes.

Selenoproteins contain the essential trace element selenium whose deficiency leads to major disorders including cancer, male reproductive system failure, or autoimmune thyroid disease. Up to now, 25 selenoprotein-encoding genes were identified in mammals, but the spatiotemporal distribution, regulation, and function of some of these selenium-containing proteins remain poorly documented. Here, we found that selenoprotein T (SelT), a new thioredoxin-like protein, is regulated by the trophic neuropeptide pituitary adenylate cyclase-activating polypeptide (PACAP) in differentiating but not mature adrenomedullary cells. In fact, our analysis revealed that, in rat, SelT is highly expressed in most embryonic structures, and then its levels decreased progressively as these organs develop, to vanish in most adult tissues. In the brain, SelT was abundantly expressed in neural progenitors in various regions such as the cortex and cerebellum but was undetectable in adult nervous cells except rostral migratory-stream astrocytes and Bergmann cells. In contrast, SelT expression was maintained in several adult endocrine tissues such as pituitary, thyroid, or testis. In the pituitary gland, SelT was found in secretory cells of the anterior lobe, whereas in the testis, the selenoprotein was present only in spermatogenic and Leydig cells. Finally, we found that SelT expression is strongly stimulated in liver cells during the regenerative process that occurs after partial hepatectomy. Taken together, these data show that SelT induction is associated with ontogenesis, tissue maturation, and regenerative mechanisms, indicating that this PACAP-regulated selenoprotein may play a crucial role in cell growth and activity in nervous, endocrine, and metabolic tissues.

Chromogranin A as a crucial factor in the sorting of peptide hormones to secretory granules.

Chromogranin A (CgA) is a soluble glycoprotein stored along with hormones and neuropeptides in secretory granules of endocrine cells. In the last four decades, intense efforts have been concentrated to characterize the structure and the biological function of CgA. Besides, CgA has been widely used as a diagnostic marker for tumors of endocrine origin, essential hypertension, various inflammatory diseases, and neurodegenerative disorders such as amyotrophic lateral sclerosis and Alzheimer's disease. CgA displays peculiar structural features, including numerous multibasic cleavage sites for prohormone convertases as well as a high proportion of acidic residues. Thus, it has been proposed that CgA represents a precursor of biologically active peptides, and a "granulogenic protein" that plays an important role as a chaperone for catecholamine storage in adrenal chromaffin cells. The widespread distribution of CgA throughout the neuroendocrine system prompted several groups to investigate the role of CgA in peptide hormone sorting to the regulated secretory pathway. This review summarizes the findings and theoretical concepts around the molecular machinery used by CgA to exert this putative intracellular function. Since CgA terminal regions exhibited strong sequence conservation through evolution, our work focused on the implication of these domains as potential functional determinants of CgA. Characterization of the molecular signals implicating CgA in the intracellular traffic of hormones represents a major biological issue that may contribute to unraveling the mechanisms defining the secretory competence of neuroendocrine cells.

Pro-hormone secretogranin II regulates dense core secretory granule biogenesis in catecholaminergic cells.

Processes underlying the formation of dense core secretory granules (DCGs) of neuroendocrine cells are poorly understood. Here, we present evidence that DCG biogenesis is dependent on the secretory protein secretogranin (Sg) II, a member of the granin family of pro-hormone cargo of DCGs in neuroendocrine cells. Depletion of SgII expression in PC12 cells leads to a decrease in both the number and size of DCGs and impairs DCG trafficking of other regulated hormones. Expression of SgII fusion proteins in a secretory-deficient PC12 variant rescues a regulated secretory pathway. SgII-containing dense core vesicles share morphological and physical properties with bona fide DCGs, are competent for regulated exocytosis, and maintain an acidic luminal pH through the V-type H(+)-translocating ATPase. The granulogenic activity of SgII requires a pH gradient along this secretory pathway. We conclude that SgII is a critical factor for the regulation of DCG biogenesis in neuroendocrine cells, mediating the formation of functional DCGs via its pH-dependent aggregation at the trans-Golgi network.

Chromogranin A promotes peptide hormone sorting to mobile granules in constitutively and regulated secreting cells: role of conserved N- and C-terminal peptides.

Chromogranin A (CgA) has been proposed to play a major role in the formation of dense-core secretory granules (DCGs) in neuroendocrine cells. Here, we took advantage of unique features of the frog CgA (fCgA) to assess the role of this granin and its potential functional determinants in hormone sorting during DCG biogenesis. Expression of fCgA in the constitutively secreting COS-7 cells induced the formation of mobile vesicular structures, which contained cotransfected peptide hormones. The fCgA and the hormones coexpressed in the newly formed vesicles could be released in a regulated manner. The N- and C-terminal regions of fCgA, which exhibit remarkable sequence conservation with their mammalian counterparts were found to be essential for the formation of the mobile DCG-like structures in COS-7 cells. Expression of fCgA in the corticotrope AtT20 cells increased pro-opiomelanocortin levels in DCGs, whereas the expression of N- and C-terminal deletion mutants provoked retention of the hormone in the Golgi area. Furthermore, fCgA, but not its truncated forms, promoted pro-opiomelanocortin sorting to the regulated secretory pathway. These data demonstrate that CgA has the intrinsic capacity to induce the formation of mobile secretory granules and to promote the sorting and release of peptide hormones. The conserved terminal peptides are instrumental for these activities of CgA.