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Stem cells in stratified epithelia are generally believed to adhere to a non-hierarchical single-progenitor model. Using lineage tracing and genetic label-retention assays, we show that the hard palatal epithelium of the oral cavity is unique in displaying marked proliferative heterogeneity. We identify a previously uncharacterized, infrequently-dividing stem cell population that resides within a candidate niche, the junctional zone (JZ). JZ stem cells tend to self-renew by planar symmetric divisions, respond to masticatory stresses, and promote wound healing, whereas frequently-dividing cells reside outside the JZ, preferentially renew through perpendicular asymmetric divisions, and are less responsive to injury. LRIG1 is enriched in the infrequently-dividing population in homeostasis, dynamically changes expression in response to tissue stresses, and promotes quiescence, whereas Igfbp5 preferentially labels a rapidly-growing, differentiation-prone population. These studies establish the oral mucosa as an important model system to study epithelial stem cell populations and how they respond to tissue stresses.
Copyright © 2019 Elsevier Inc. All rights reserved.
Successful separation of two daughter cells (i.e., cytokinesis) is essential for life. Many eukaryotic cells divide using a contractile apparatus called the cytokinetic ring (CR) that associates dynamically with the plasma membrane (PM) and generates force that contributes to PM ingression between daughter cells. In important membrane-CR scaffolds include the paralogous F-BAR proteins Cdc15 and Imp2. Their conserved protein structure consists of the archetypal F-BAR domain linked to an SH3 domain by an intrinsically disordered region (IDR). Functions have been assigned to the F-BAR and SH3 domains. In this study we probed the function of the central IDR. We found that the IDR of Cdc15 is essential for viability and cannot be replaced by that of Imp2, whereas the F-BAR domain of Cdc15 can be swapped with several different F-BAR domains, including that of Imp2. Deleting part of the IDR results in CR defects and abolishes calcineurin phosphatase localization to the CR. Together these results indicate that Cdc15's IDR has a nonredundant essential function that coordinates regulation of CR architecture.
Animal cells, amoebas and yeast divide using a force-generating, actin- and myosin-based contractile ring or 'cytokinetic ring' (CR). Despite intensive research, questions remain about the spatial organization of CR components, the mechanism by which the CR generates force, and how other cellular processes are coordinated with the CR for successful membrane ingression and ultimate cell separation. This Review highlights new findings about the spatial relationship of the CR to the plasma membrane and the arrangement of molecules within the CR from studies using advanced microscopy techniques, as well as mechanistic information obtained from approaches. We also consider advances in understanding coordinated cellular processes that impact the architecture and function of the CR.
© 2019. Published by The Company of Biologists Ltd.
Neuronal-glial relationships play a critical role in the maintenance of central nervous system architecture and neuronal specification. A deeper understanding of these relationships can elucidate cellular cross-talk capable of sustaining proper development of neural tissues. In the cerebellum, cerebellar granule neuron precursors (CGNPs) proliferate in response to Purkinje neuron-derived Sonic hedgehog (Shh) before ultimately exiting the cell cycle and migrating radially along Bergmann glial fibers. However, the function of Bergmann glia in CGNP proliferation remains not well defined. Interestingly, the Hh pathway is also activated in Bergmann glia, but the role of Shh signaling in these cells is unknown. In this study, we show that specific ablation of Shh signaling using the tamoxifen-inducible TNC line to eliminate Shh pathway activator Smoothened in Bergmann glia is sufficient to cause severe cerebellar hypoplasia and a significant reduction in CGNP proliferation. TNC; Smo (Smo) mice demonstrate an obvious reduction in cerebellar size within two days of ablation of Shh signaling. Mutant cerebella have severely reduced proliferation and increased differentiation of CGNPs due to a significant decrease in Shh activity and concomitant activation of Wnt signaling in Smo CGNPs, suggesting that this pathway is involved in cross-talk with the Shh pathway in regulating CGNP proliferation. In addition, Purkinje cells are ectopically located, their dendrites stunted, and the Bergmann glial network disorganized. Collectively, these data demonstrate a previously unappreciated role for Bergmann glial Shh signaling activity in the proliferation of CGNPs and proper maintenance of cerebellar architecture.
Copyright © 2018 Elsevier Inc. All rights reserved.
In , cytokinesis requires the assembly and constriction of an actomyosin-based contractile ring (CR). A single essential formin, Cdc12, localizes to the cell middle upon mitotic onset and nucleates the F-actin of the CR. Cdc12 medial recruitment is mediated in part by its direct binding to the F-BAR scaffold Cdc15. Given that Cdc12 is hyperphosphorylated in M phase, we explored whether Cdc12 phosphoregulation impacts its association with Cdc15 during mitosis. We found that Cdk1, a major mitotic kinase, phosphorylates Cdc12 on six N-terminal residues near the Cdc15-binding site, and phosphorylation on these sites inhibits its interaction with the Cdc15 F-BAR domain. Consistent with this finding, a mutant with all six Cdk1 sites changed to phosphomimetic residues () displays phenotypes similar to , in which the Cdc15-binding motif is disrupted; both show reduced Cdc12 at the CR and delayed CR formation. Together, these results indicate that Cdk1 phosphorylation of formin Cdc12 antagonizes its interaction with Cdc15 and thereby opposes Cdc12's CR localization. These results are consistent with a general role for Cdk1 in inhibiting cytokinesis until chromosome segregation is complete.
© 2018 Willet et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).
Cerebellar growth and foliation require the Hedgehog-driven proliferation of granule cell precursors (GCPs) in the external granule layer (EGL). However, that increased or extended GCP proliferation generally does not elicit ectopic folds suggests that additional determinants control cortical expansion and foliation during cerebellar development. Here, we find that genetic loss of the serine-threonine kinase Liver Kinase B1 (Lkb1) in GCPs increased cerebellar cortical size and foliation independent of changes in proliferation or Hedgehog signaling. This finding is unexpected given that Lkb1 has previously shown to be critical for Hedgehog pathway activation in cultured cells. Consistent with unchanged proliferation rate of GCPs, the cortical expansion of Lkb1 mutants is accompanied by thinning of the EGL. The plane of cell division, which has been implicated in diverse processes from epithelial surface expansions to gyrification of the human cortex, remains unchanged in the mutants when compared to wild-type controls. However, we find that Lkb1 mutants display delayed radial migration of post-mitotic GCPs that coincides with increased cortical size, suggesting that aberrant cell migration may contribute to the cortical expansion and increase foliation. Taken together, our results reveal an important role for Lkb1 in regulating cerebellar cortical size and foliation in a Hedgehog-independent manner.
Copyright © 2017 Elsevier Inc. All rights reserved.
The contractile ring is a complex molecular apparatus which physically divides many eukaryotic cells. Despite knowledge of its protein composition, the molecular architecture of the ring is not known. Here we have applied super-resolution microscopy and FRET to determine the nanoscale spatial organization of contractile ring components relative to the plasma membrane. Similar to other membrane-tethered actin structures, we find proteins localize in specific layers relative to the membrane. The most membrane-proximal layer (0-80 nm) is composed of membrane-binding scaffolds, formin, and the tail of the essential myosin-II. An intermediate layer (80-160 nm) consists of a network of cytokinesis accessory proteins as well as multiple signaling components which influence cell division. Farthest from the membrane (160-350 nm) we find F-actin, the motor domains of myosins, and a major F-actin crosslinker. Circumferentially within the ring, multiple proteins proximal to the membrane form clusters of different sizes, while components farther from the membrane are uniformly distributed. This comprehensive organizational map provides a framework for understanding contractile ring function.
Nuclear pore complexes (NPCs), which are composed of nucleoporins (Nups) and regulate transport between the nucleus and cytoplasm, significantly impact the replicative life span (RLS) of We previously reported that deletion of the nonessential gene increases RLS, although the molecular basis for this effect was unknown. In this study, we find that nuclear tRNA accumulation contributes to increased longevity in Δ cells. Fluorescence in situ hybridization (FISH) experiments demonstrate that several specific tRNAs accumulate in the nuclei of Δ mutants. Protein levels of the transcription factor Gcn4 are increased when is deleted, and is required for the elevated life spans of Δ mutants, similar to other previously described tRNA export and ribosomal mutants. Northern blots indicate that tRNA splicing and aminoacylation are not significantly affected in Δ cells, suggesting that Nup100 is largely required for nuclear export of mature, processed tRNAs. Distinct tRNAs accumulate in the nuclei of Δ and Δ mutants, while Los1-GFP nucleocytoplasmic shuttling is unaffected by Nup100. Thus, we conclude that Nup100 regulates tRNA export in a manner distinct from Los1 or Msn5. Together, these experiments reveal a novel Nup100 role in the tRNA life cycle that impacts the life span.
© 2017 Lord et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.
The final steps of cell division are tightly coordinated in space and time, but whether mechanisms exist to couple the actin and microtubule (MT) cytoskeletons during anaphase and cytokinesis (C phase) is largely unknown. During anaphase, MTs are incorporated into an anti-parallel array termed the spindle midzone (midzone MTs), whereas F-actin and non-muscle myosin II, together with other factors, organize into the cleavage furrow . Previous studies in somatic cells have shown that midzone MTs become highly stable after furrows have begun ingression , indicating that furrow-to-MT communication may occur. Midzone formation is also inhibited in fly spermatocytes that fail to form a cleavage furrow  and during monopolar cytokinesis when myosin contractility is blocked by blebbistatin . We show here that midzone MT stabilization is dependent on actomyosin contraction, suggesting that there is active coordination between furrow ingression and microtubule dynamics. Midzone microtubule stabilization also depends on the kinase activity of Aurora B, the catalytic subunit of the chromosomal passenger complex (CPC), uncovering a feedback mechanism that couples furrowing with microtubule dynamics. We further show that the CPC scaffolding protein INCENP (inner centromere protein) binds actin, an interaction that is important for cytokinesis and for midzone MT stabilization following furrow ingression. Stabilization of midzone MTs with low amounts of Taxol rescues cytokinesis in INCENP actin-binding mutant-expressing cells. Collectively, our work demonstrates that the actin and microtubule cytoskeletons are coordinated during cytokinesis and suggests that the CPC is integral for coupling furrow ingression with midzone microtubule stabilization.
Copyright © 2016 Elsevier Ltd. All rights reserved.
Division site positioning is critical for both symmetric and asymmetric cell divisions. In many organisms, positive and negative signals cooperate to position the contractile actin ring for cytokinesis. In rod-shaped fission yeast Schizosaccharomyces pombe cells, division at midcell is achieved through positive Mid1/anillin-dependent signaling emanating from the central nucleus and negative signals from the dual-specificity tyrosine phosphorylation-regulated kinase family kinase Pom1 at the cell poles. In this study, we show that Pom1 directly phosphorylates the F-BAR protein Cdc15, a central component of the cytokinetic ring. Pom1-dependent phosphorylation blocks Cdc15 binding to paxillin Pxl1 and C2 domain protein Fic1 and enhances Cdc15 dynamics. This promotes ring sliding from cell poles, which prevents septum assembly at the ends of cells with a displaced nucleus or lacking Mid1. Pom1 also slows down ring constriction. These results indicate that a strong negative signal from the Pom1 kinase at cell poles converts Cdc15 to its closed state, destabilizes the actomyosin ring, and thus promotes medial septation.
© 2015 Ullal et al.