Stem Cells and Niches: Mechanisms That Promote Stem Cell Maintenance throughout Life

Mechanisms of Asymmetric Stem Cell Division

Many genes and molecules that drive tissue patterning during organogenesis and tissue regeneration have been discovered. Yet, we still lack a full understanding of how these chemical cues induce the formation of living tissues with their unique shapes and material properties. Here, we review work based on the convergence of physics, engineering and biology that suggests that mechanical forces generated by living cells are as crucial as genes and chemical signals for the control of embryological development, morphogenesis and tissue patterning.

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Mechanical control of tissue and organ development

The PAR Proteins: Fundamental Players in Animal Cell Polarization

There is currently an unmet need for the supply of autologous, patient-specific stem cells for regenerative therapies in the clinic. Mesenchymal stem cell differentiation can be driven by the material/cell interface suggesting a unique strategy to manipulate stem cells in the absence of complex soluble chemistries or cellular reprogramming. However, so far the derivation and identification of surfaces that allow retention of multipotency of this key regenerative cell type have remained elusive. Adult stem cells spontaneously differentiate in culture, resulting in a rapid diminution of the multipotent cell population and their regenerative capacity. Here we identify a nanostructured surface that retains stem-cell phenotype and maintains stem-cell growth over eight weeks. Furthermore, the study implicates a role for small RNAs in repressing key cell signalling and metabolomic pathways, demonstrating the potential of surfaces as non-invasive tools with which to address the stem cell niche.

Nanoscale surfaces for the long-term maintenance of mesenchymal stem cell phenotype and multipotency

Development of a multicellular organism from a fertilized egg depends on a precise balance between symmetric cell divisions to expand the pool of similar cells, and asymmetric cell divisions to create cell-type diversity. Spindle orientation can influence the generation of symmetric or asymmetric cell fates depending on how it is coupled to cell-intrinsic polarity cues, or how it is positioned relative to cell-extrinsic cues such as niche-derived signals. In this review, we describe the mechanism of spindle orientation in budding yeast, Drosophila melanogaster, Caenorhabditis elegans and mammalian neural progenitors, with the goal of highlighting conserved mechanisms and indicating open questions for the future.

http://uoneuro.uoregon.edu/doelab/pdfs2/siller09.pdf

Spindle orientation during asymmetric cell division.

Asymmetric cell division generates cell diversity during development and regulates stem-cell self-renewal in Drosophila and mammals. In Drosophila, neuroblasts align their spindle with a cortical Partner of Inscuteable (Pins)-G alpha i crescent to divide asymmetrically, but the link between cortical polarity and the mitotic spindle is poorly understood. Here, we show that Pins directly binds, and coimmunoprecipitates with, the NuMA-related Mushroom body defect (Mud) protein. Pins recruits Mud to the neuroblast apical cortex, and Mud is also strongly localized to centrosome/spindle poles, in a similar way to NuMA. In mud mutants, cortical polarity is normal, but the metaphase spindle frequently fails to align with the cortical polarity axis. When spindle orientation is orthogonal to cell polarity, symmetric division occurs. We propose that Mud is a functional orthologue of mammalian NuMA and Caenorhabditis elegans Lin-5, and that Mud coordinates spindle orientation with cortical polarity to promote asymmetric cell division.

/pdf/the-numa-related-mud-protein-binds-pins-and-regulates-vhqoyxi0am.pdf

The NuMA-related Mud protein binds Pins and regulates spindle orientation in Drosophila neuroblasts.

Lis1 is required for nuclear migration in fungi, cell cycle progression in mammals, and the formation of a folded cerebral cortex in humans. Lis1 binds dynactin and the dynein motor complex, but the role of Lis1 in many dynein/dynactin-dependent processes is not clearly understood. Here we generate and/or characterize mutants for Drosophila Lis1 and a dynactin subunit, Glued, to investigate the role of Lis1/dynactin in mitotic checkpoint function. In addition, we develop an improved time-lapse video microscopy technique that allows live imaging of GFP-Lis1, GFP-Rod checkpoint protein, green fluorescent protein (GFP)-labeled chromosomes, or GFP-labeled mitotic spindle dynamics in neuroblasts within whole larval brain explants. Our mutant analyses show that Lis1/dynactin have at least two independent functions during mitosis: first promoting centrosome separation and bipolar spindle assembly during prophase/prometaphase, and subsequently generating interkinetochore tension and transporting checkpoint proteins off kinetochores during metaphase, thus promoting timely anaphase onset. Furthermore, we show that Lis1/dynactin/dynein physically associate and colocalize on centrosomes, spindle MTs, and kinetochores, and that regulation of Lis1/dynactin kinetochore localization in Drosophila differs from both Caenorhabditis elegans and mammals. We conclude that Lis1/dynactin act together to regulate multiple, independent functions in mitotic cells, including spindle formation and cell cycle checkpoint release.

/pdf/live-imaging-of-drosophila-brain-neuroblasts-reveals-a-role-4iifpntw61.pdf

Live Imaging of Drosophila Brain Neuroblasts Reveals a Role for Lis1/Dynactin in Spindle Assembly and Mitotic Checkpoint Control

http://uoneuro.uoregon.edu/doelab/pdfs2/Siller08.pdf

Lis1/dynactin regulates metaphase spindle orientation in Drosophila neuroblasts.

Drosophila neuroblasts divide asymmetrically by aligning their mitotic spindle with cortical cell polarity to generate distinct sibling cell types. Neuroblasts asymmetrically localize Gαi, Pins, and Mud proteins; Pins/Gαi direct cortical polarity, whereas Mud is required for spindle orientation. It is currently unknown how Gαi–Pins–Mud binding is regulated to link cortical polarity with spindle orientation. Here, we show that Pins forms a “closed” state via intramolecular GoLoco–tetratricopeptide repeat (TPR) interactions, which regulate Mud binding. Biochemical, genetic, and live imaging experiments show that Gαi binds to the first of three Pins GoLoco motifs to recruit Pins to the apical cortex without “opening” Pins or recruiting Mud. However, Gαi and Mud bind cooperatively to the Pins GoLocos 2/3 and tetratricopeptide repeat domains, respectively, thereby restricting Pins–Mud interaction to the apical cortex and fixing spindle orientation. We conclude that Pins has multiple activity states that generate cortical polarity and link it with mitotic spindle orientation.

https://www.pnas.org/content/pnas/104/36/14306.full.pdf

Karsten H. Siller

Papers

Spindle orientation during asymmetric cell division.

The NuMA-related Mud protein binds Pins and regulates spindle orientation in Drosophila neuroblasts.

Live Imaging of Drosophila Brain Neuroblasts Reveals a Role for Lis1/Dynactin in Spindle Assembly and Mitotic Checkpoint Control

Lis1/dynactin regulates metaphase spindle orientation in Drosophila neuroblasts.

Gαi generates multiple Pins activation states to link cortical polarity and spindle orientation in Drosophila neuroblasts