Nuclear envelope assembly and dynamics during development.

The nuclear envelope (NE) protects but also organizes the eukaryotic genome. In this review we will discuss recent literature on how the NE disassembles and reassembles, how it varies in surface area and protein composition and how this translates into chromatin organization and gene expression in the context of animal development.

Co-translational assembly orchestrates competing biogenesis pathways.

During the co-translational assembly of protein complexes, a fully synthesized subunit engages with the nascent chain of a newly synthesized interaction partner. Such events are thought to contribute to productive assembly, but their exact physiological relevance remains underexplored. Here, we examine structural motifs contained in nucleoporins for their potential to facilitate co-translational assembly. We experimentally test candidate structural motifs and identify several previously unknown co-translational interactions. We demonstrate by selective ribosome profiling that domain invasion motifs of beta-propellers, coiled-coils, and short linear motifs may act as co-translational assembly domains. Such motifs are often contained in proteins that are members of multiple complexes (moonlighters) and engage with closely related paralogs. Surprisingly, moonlighters and paralogs assemble co-translationally in only some but not all of the relevant biogenesis pathways. Our results highlight the regulatory complexity of assembly pathways.

A modular platform for automated cryo-FIB workflows.

Lamella micromachining by focused ion beam milling at cryogenic temperature (cryo-FIB) has matured into a preparation method widely used for cellular cryo-electron tomography. Due to the limited ablation rates of low Ga+ ion beam currents required to maintain the structural integrity of vitreous specimens, common preparation protocols are time-consuming and labor intensive. The improved stability of new-generation cryo-FIB instruments now enables automated operations. Here, we present an open-source software tool, SerialFIB, for creating automated and customizable cryo-FIB preparation protocols. The software encompasses a graphical user interface for easy execution of routine lamellae preparations, a scripting module compatible with available Python packages, and interfaces with three-dimensional correlative light and electron microscopy (CLEM) tools. SerialFIB enables the streamlining of advanced cryo-FIB protocols such as multi-modal imaging, CLEM-guided lamella preparation and in situ lamella lift-out procedures. Our software therefore provides a foundation for further development of advanced cryogenic imaging and sample preparation protocols.

Lis1 activates dynein motility by modulating its pairing with dynactin.

Lissencephaly-1 (Lis1) is a key cofactor for dynein-mediated intracellular transport towards the minus-ends of microtubules. It remains unclear whether Lis1 serves as an inhibitor or an activator of mammalian dynein motility. Here we use single-molecule imaging and optical trapping to show that Lis1 does not directly alter the stepping and force production of individual dynein motors assembled with dynactin and a cargo adaptor. Instead, Lis1 promotes the formation of an active complex with dynactin. Lis1 also favours the recruitment of two dyneins to dynactin, resulting in increased velocity, higher force production and more effective competition against kinesin in a tug-of-war. Lis1 dissociates from motile complexes, indicating that its primary role is to orchestrate the assembly of the transport machinery. We propose that Lis1 binding releases dynein from its autoinhibited state, which provides a mechanistic explanation for why Lis1 is required for efficient transport of many dynein-associated cargos in cells.

Evidence that a positive feedback loop drives centrosome maturation in fly embryos.

Centrosomes are formed when mother centrioles recruit pericentriolar material (PCM) around themselves. The PCM expands dramatically as cells prepare to enter mitosis (a process termed centrosome maturation), but it is unclear how this expansion is achieved. In flies, Spd-2 and Cnn are thought to form a scaffold around the mother centriole that recruits other components of the mitotic PCM, and the Polo-dependent phosphorylation of Cnn at the centrosome is crucial for scaffold assembly. Here, we show that, like Cnn, Spd-2 is specifically phosphorylated at centrosomes. This phosphorylation appears to create multiple phosphorylated S-S/T(p) motifs that allow Spd-2 to recruit Polo to the expanding scaffold. If the ability of Spd-2 to recruit Polo is impaired, the scaffold is initially assembled around the mother centriole, but it cannot expand outwards, and centrosome maturation fails. Our findings suggest that interactions between Spd-2, Polo and Cnn form a positive feedback loop that drives the dramatic expansion of the mitotic PCM in fly embryos.

A homeostatic clock sets daughter centriole size in flies.

Centrioles are highly structured organelles whose size is remarkably consistent within any given cell type. New centrioles are born when Polo-like kinase 4 (Plk4) recruits Ana2/STIL and Sas-6 to the side of an existing "mother" centriole. These two proteins then assemble into a cartwheel, which grows outwards to form the structural core of a new daughter. Here, we show that in early Drosophila melanogaster embryos, daughter centrioles grow at a linear rate during early S-phase and abruptly stop growing when they reach their correct size in mid- to late S-phase. Unexpectedly, the cartwheel grows from its proximal end, and Plk4 determines both the rate and period of centriole growth: the more active the centriolar Plk4, the faster centrioles grow, but the faster centriolar Plk4 is inactivated and growth ceases. Thus, Plk4 functions as a homeostatic clock, establishing an inverse relationship between growth rate and period to ensure that daughter centrioles grow to the correct size.

Lissencephaly-1 is a context-dependent regulator of the human dynein complex.

The cytoplasmic dynein-1 (dynein) motor plays a central role in microtubule organisation and cargo transport. These functions are spatially regulated by association of dynein and its accessory complex dynactin with dynamic microtubule plus ends. Here, we elucidate in vitro the roles of dynactin, end-binding protein-1 (EB1) and Lissencephaly-1 (LIS1) in the interaction of end tracking and minus end-directed human dynein complexes with these sites. LIS1 promotes dynactin-dependent tracking of dynein on both growing and shrinking plus ends. LIS1 also increases the frequency and velocity of processive dynein movements that are activated by complex formation with dynactin and a cargo adaptor. This stimulatory effect of LIS1 contrasts sharply with its documented ability to inhibit the activity of isolated dyneins. Collectively, our findings shed light on how mammalian dynein complexes associate with dynamic microtubules and help clarify how LIS1 promotes the plus-end localisation and cargo transport functions of dynein in vivo.

Dissecting the function and assembly of acentriolar microtubule organizing centers in Drosophila cells in vivo.

Acentriolar microtubule organizing centers (aMTOCs) are formed during meiosis and mitosis in several cell types, but their function and assembly mechanism is unclear. Importantly, aMTOCs can be overactive in cancer cells, enhancing multipolar spindle formation, merotelic kinetochore attachment and aneuploidy. Here we show that aMTOCs can form in acentriolar Drosophila somatic cells in vivo via an assembly pathway that depends on Asl, Cnn and, to a lesser extent, Spd-2--the same proteins that appear to drive mitotic centrosome assembly in flies. This finding enabled us to ablate aMTOC formation in acentriolar cells, and so perform a detailed genetic analysis of the contribution of aMTOCs to acentriolar mitotic spindle formation. Here we show that although aMTOCs can nucleate microtubules, they do not detectably increase the efficiency of acentriolar spindle assembly in somatic fly cells. We find that they are required, however, for robust microtubule array assembly in cells without centrioles that also lack microtubule nucleation from around the chromatin. Importantly, aMTOCs are also essential for dynein-dependent acentriolar spindle pole focusing and for robust cell proliferation in the absence of centrioles and HSET/Ncd (a kinesin essential for acentriolar spindle pole focusing in many systems). We propose an updated model for acentriolar spindle pole coalescence by the molecular motors Ncd/HSET and dynein in conjunction with aMTOCs.

The centrosome-specific phosphorylation of Cnn by Polo/Plk1 drives Cnn scaffold assembly and centrosome maturation.

Centrosomes are important cell organizers. They consist of a pair of centrioles surrounded by pericentriolar material (PCM) that expands dramatically during mitosis-a process termed centrosome maturation. How centrosomes mature remains mysterious. Here, we identify a domain in Drosophila Cnn that appears to be phosphorylated by Polo/Plk1 specifically at centrosomes during mitosis. The phosphorylation promotes the assembly of a Cnn scaffold around the centrioles that is in constant flux, with Cnn molecules recruited continuously around the centrioles as the scaffold spreads slowly outward. Mutations that block Cnn phosphorylation strongly inhibit scaffold assembly and centrosome maturation, whereas phosphomimicking mutations allow Cnn to multimerize in vitro and to spontaneously form cytoplasmic scaffolds in vivo that organize microtubules independently of centrosomes. We conclude that Polo/Plk1 initiates the phosphorylation-dependent assembly of a Cnn scaffold around centrioles that is essential for efficient centrosome maturation in flies.

Centrosome loss or amplification does not dramatically perturb global gene expression in Drosophila.

Centrosome defects are a common feature of many cancers, and they can predispose fly brain cells to form tumours. In flies, centrosome defects perturb the asymmetric division of the neural stem cells, but it is unclear how this might lead to malignant transformation. One possibility is that centrosome defects might also perturb cellular homeostasis: for example, stress pathways are often activated in response to centrosome defects in cultured cells, and stress contributes to tumourigenesis in some fly models. Here we attempt to assess whether centrosome loss or centrosome amplification perturbs cell physiology in vivo by profiling the global transcriptome of Drosophila larval brains and imaginal discs that either lack centrosomes or have too many centrosomes. Surprisingly, we find that centrosome loss or amplification leads to few changes in the transcriptional profile of these cells, indicating that centrosome defects are surprisingly well tolerated by these cells. These observations indicate that centrosome defects can predispose fly brain cells to form tumours without, at least initially, dramatically altering their physiology.

Short-term starvation of immune deficient Drosophila improves survival to gram-negative bacterial infections.

BACKGROUND: Primary immunodeficiencies are inborn errors of immunity that lead to life threatening conditions. These predispositions describe human immunity in natura and highlight the important function of components of the Toll-IL-1- receptor-nuclear factor kappa B (TIR-NF-kappaB) pathway. Since the TIR-NF-kappaB circuit is a conserved component of the host defence in higher animals, genetically tractable models may contribute ideas for clinical interventions. METHODOLOGY/PRINCIPAL FINDINGS: We used immunodeficient fruit flies (Drosophila melanogaster) to address questions pertaining to survival following bacterial infection. We describe here that flies lacking the NF-kappaB protein Relish, indispensable for countering Gram-negative bacteria, had a greatly improved survival to such infections when subject to dietary short-term starvation (STS) prior to immune challenge. STS induced the release of Nitric Oxide (NO), a potent molecule against pathogens in flies, mice and humans. Administering the NO Synthase-inhibitory arginine analog N-Nitro-L-Arginine-Methyl-Ester (L-NAME) but not its inactive enantiomer D-NAME increased once again sensitivity to infection to levels expected for relish mutants. Surprisingly, NO signalling required the NF-kappaB protein Dif, usually needed for responses against Gram-positive bacteria. CONCLUSIONS/SIGNIFICANCE: Our results show that NO release through STS may reflect an evolutionary conserved process. Moreover, STS could be explored to address immune phenotypes related to infection and may offer ways to boost natural immunity.