Jens Januschke

The size of cells varies enormously in biology and how cell size and function are linked are fundamental questions. Asymmetrically dividing Drosophila neural stem cells, also called neuroblasts, produce a large neuroblast and a smaller ganglion mother cell (GMC) at each division. However, why GMCs need to be smaller than neuroblasts has largely remained unexplained. Here, we developed acute and reversible methods to alter daughter-cell size during a single neuroblast division while preserving cortical polarity and asymmetric inheritance of fate determinants. We identify atypical protein kinase C (aPKC) as a regulator of size asymmetry and show that transient and partial co-inhibition of aPKC and Rho kinase (Drok in Drosophila) generates daughters spanning a size range, including size-wise near-symmetric sibling cells while polarity is preserved. We find that above a certain size threshold daughter cells retain neuroblast-like properties despite inheriting GMC determinants: they divide repeatedly, exhibit neuroblast-like cell-cycle timing, and undergo size-wise asymmetric division. Smaller daughters below this threshold instead follow a GMC-like trajectory. Thus, in Drosophila type I neuroblasts, daughter-cell size can act as a regulator of lineage behaviour, overriding inherited fate cues in sufficiently large daughter cells.

Directionality of protein traffic during secretion is driven by sorting receptors that bind cargo in one compartment and release them in another. Erv14/cornichon family receptors mediate delivery from the endoplasmic reticulum (ER) to the Golgi. Erv14 traffics its client, Mid2, via a conserved interface formed by two transmembrane helices (TMHs). Molecular dynamics simulations of Erv14-Mid2 suggest that Erv14 TMHs undergo dynamic movement in response to both client binding and lipid bilayer thickness. Interaction between charged residues that flank the TMHs of Mid2 and Erv14 couple client binding to stabilization of the Erv14 export signal and thus engagement with the COPII coat. We term this mechanism Helix Engagement Induced Signal-mediated Traffic (HEIST) and suggest it as a general mechanism where changes in membrane biophysical properties drive directional transport.

The apicomedial actomyosin network is crucial for generating mechanical forces in cells. Oscillatory behavior of this contractile network is commonly observed before or during significant morphogenetic events. For instance, during the development of the Drosophila adult abdominal epidermis, larval epithelial cells (LECs) undergo pulsed contractions before being replaced by histoblasts. These contractions involve the formation of contracted regions of concentrated actin and myosin. The emergence and control of pulsed contractions are not fully understood. Here, we combined in vivo 4D microscopy with numerical simulations of an active elastomer model applied to realistic cell geometries and boundary conditions informed by cell polarity to study in vivo subcellular spatial patterns of LEC actomyosin dynamics. The active elastomer model quantitatively reproduced in vivo observations. When compared to rectangular domains, simulations on realistic cell geometries showed systematically better agreement with experiments. We found that cell shape, cell polarity, and organization of the cell’s actomyosin network codetermine spatiotemporal network dynamics both in vivo and in simulations. Furthermore, the model predicted changes to LEC contractile activity under genetic perturbation of the actomyosin network. Our results show that cell geometry, accompanied by boundary conditions which reflect the cells’ polarity, is important to understanding the dynamics of the apicomedial actomyosin network. Moreover, our findings support the notion that spatiotemporal oscillatory behavior of the actomyosin network is an emergent property of the actomyosin network, rather than driven by upstream signaling.

Investigation of cell behaviour and cell biological processes underlying human development is facilitated by the creation of fluorescent reporters in human pluripotent stem cells, which can be differentiated into cell types of choice. Here, we report use of a PiggyBac transposon-mediated stable integration strategy to engineer human pluripotent stem cell reporter lines. These express a plasma membrane-localised protein tagged with the fluorescent proteins eGFP or mKate2, the photoconvertible nuclear marker H2B-mEos3.2, or the cytoskeletal protein F-Tractin tagged with mKate2. Focussing on neural development, these lines were used to live image and quantify cell behaviours, including cell cycle progression and cell division orientation in spinal cord rosettes. Further, lipofection-mediated introduction of PiggyBac constructs into human neural progenitors labelled single cells and small cell groups within rosettes, allowing individual cell behaviours including neuronal delamination to be monitored. Finally, using the F-Tractin-mKate2 hiPSC line, actin dynamics were captured during proliferation in cortical neural rosettes. This study presents and validates new tools and techniques with which to interrogate human cell behaviour and cell biology using live-imaging approaches.

The Actomyosin cortex is a highly dynamic part of the cytoskeleton and cells can modulate its properties. This is important for essential cellular functions including migration, cell shape control and cleavage furrow positioning during cell division. Asymmetrically dividing Drosophila neural stem cells, called neuroblasts, exhibit stereotypic patterns of Myosin dynamics along the cell cycle. Some of the Myosin dynamics manifest as flows and are important to asymmetrically position the cleavage furrow which establishes the cell size-difference between the neuroblast and its daughter cell. Myosin can be regulated by small GTPases and their GAPs and GEFs, providing spatio-temporal control. Rho kinase downstream of the small GTPase Rho regulates Myosin activity, yet the regulation and contribution of its GEFs and GAPs to asymmetric neuroblast division are not resolved. Here, we systematically analysed the localization of RhoGAPs and RhoGEFs expressed in neuroblasts and identified the ARHGAP19 homolog RhoGAP54D as a regulator of Myosin dynamics. A cytoplasmic pool of RhoGAP54D suppresses pulsatile Myosin activity during interphase and metaphase, regulated by the nucleoporin Members Only via slow nuclear import. In anaphase RhoGAP54D is recruited to the apical cortex and constitutes the trigger that initiates the directional Myosin flow that positions the cleavage furrow. This depends on PsGEF which is itself recruited downstream of Par3/Baz and Pins, linking cell polarity to RhoGAP54D activity to promote daughter cell size asymmetry during neuroblast division.

The apicomedial actomyosin network is crucial for generating mechanical forces in cells. Oscillatory behaviour of this contractile network is commonly observed before or during significant morphogenetic events. For instance, during the development of the Drosophila adult abdominal epidermis, larval epithelial cells (LECs) undergo pulsed contractions before being replaced by histoblasts. These contractions involve the formation of contracted regions of concentrated actin and myosin. However, the emergence and control of pulsed contractions are not fully understood. Here, we combined in vivo 4D microscopy with numerical simulations of an active elastomer model applied to realistic cell geometries and boundary conditions to study LEC actomyosin dynamics. The active elastomer model was able to reproduce in vivo observations quantitatively. We also characterised the relationship between cell shape, cell polarity, and actomyosin network parameters with the spatiotemporal characteristics of the contractile network both in vivo and in simulations. Our results show that cell geometry, accompanied by boundary conditions which reflect the cells’ polarity, is essential to understand the dynamics of the apicomedial actomyosin network. Moreover, our findings support the notion that spatiotemporal oscillatory behaviour of the actomyosin network is an emergent property of the actomyosin network, rather than driven by upstream signalling.

The generation of distinct cell fates during development depends on asymmetric cell division of progenitor cells. In the central and peripheral nervous system of Drosophila, progenitor cells respectively called neuroblasts or sensory organ precursors use PAR polarity during mitosis to control cell fate determination in their daughter cells. How polarity and the cell cycle are coupled, and how the cell cycle machinery regulates PAR protein function and cell fate determination is poorly understood. Here, we generate an analog sensitive allele of CDK1 and reveal that its partial inhibition weakens but does not abolish apical polarity in embryonic and larval neuroblasts and leads to defects in polarisation of fate determinants. We describe a novel in vivo phosphorylation of Bazooka, the Drosophila homolog of PAR-3, on Serine180, a consensus CDK phosphorylation site. In some tissular contexts, phosphorylation of Serine180 occurs in asymmetrically dividing cells but not in their symmetrically dividing neighbours. In neuroblasts, Serine180 phosphomutants disrupt the timing of basal polarisation. Serine180 phosphomutants also affect the specification and binary cell fate determination of sensory organ precursors as well as Baz localisation during their asymmetric cell divisions. Finally, we show that CDK1 phosphorylates Serine-S180 and an equivalent Serine on human PAR-3 in vitro.

Investigation of cell behaviour and cell biological processes underlying human development is facilitated by creation of fluorescent reporters in human pluripotent stem cells, which can be differentiated into cell types of choice. Here we report use of a piggyBac transposon-mediated stable integration strategy to engineer human pluripotent stem cell reporter lines. These express a plasma membrane localised protein tagged with the fluorescent proteins eGFP or mKate2, the photoconvertible nuclear marker H2B-mEos3.2, or the cytoskeletal protein F-tractin tagged with mKate2. Focussing on neural development these lines were used to live image and quantify cell behaviours, including cell cycle progression and cell division orientation in spinal cord rosettes. Further, lipofection-mediated introduction of piggyBac constructs into human neural progenitors labelled single cells and small cell groups within rosettes, allowing individual cell behaviours including neuronal delamination to be monitored. Finally, using the F-tractin-mKate2 hiPSC line, novel actin dynamics were captured during proliferation in cortical neural rosettes. This study presents and validates new tools and techniques with which to interrogate human cell behaviour and cell biology using live imaging approaches.