Separation-of-function MCPH-associated mutations in CPAP affect centriole number and length.

Centrioles are microtubule-based cylindrical ultrastructures characterized by their definite size and robustness. The molecular capping protein, CPAP (also known as CENPJ) engages its N-terminal region with the centriole microtubules to regulate their length. Nevertheless, the conserved C-terminal glycine-rich G-box of CPAP, which interacts with the centriole inner cartwheel protein STIL, is frequently mutated in primary microcephaly (MCPH) patients. Here, we show that two different MCPH-associated variants, E1235V and D1196N in the CPAP G-box, affect distinct functions at centrioles. The E1235V mutation reduces CPAP centriole recruitment and causes overly long centrioles. The D1196N mutation increases centriole numbers without affecting centriole localization. Both mutations prevent binding to STIL, which controls centriole duplication. Our work highlights the involvement of an alternative CEP152-dependent route for CPAP centriole localization. Molecular dynamics simulations suggest that E1235V leads to an increase in G-box flexibility, which could have implications on its molecular interactions. Collectively, we demonstrate that a CPAP region outside the microtubule-interacting domains influences centriole number and length, which translates to spindle defects and reduced cell viability. Our work provides new insights into the molecular causes of primary microcephaly.

Bendless is essential for PINK1-Park mediated Mitofusin degradation under mitochondrial stress caused by loss of LRPPRC.

Cells under mitochondrial stress often co-opt mechanisms to maintain energy homeostasis, mitochondrial quality control and cell survival. A mechanistic understanding of such responses is crucial for further insight into mitochondrial biology and diseases. Through an unbiased genetic screen in Drosophila, we identify that mutations in lrpprc2, a homolog of the human LRPPRC gene that is linked to the French-Canadian Leigh syndrome, result in PINK1-Park activation. While the PINK1-Park pathway is well known to induce mitophagy, we show that PINK1-Park regulates mitochondrial dynamics by inducing the degradation of the mitochondrial fusion protein Mitofusin/Marf in lrpprc2 mutants. In our genetic screen, we also discover that Bendless, a K63-linked E2 conjugase, is a regulator of Marf, as loss of bendless results in increased Marf levels. We show that Bendless is required for PINK1 stability, and subsequently for PINK1-Park mediated Marf degradation under physiological conditions, and in response to mitochondrial stress as seen in lrpprc2. Additionally, we show that loss of bendless in lrpprc2 mutant eyes results in photoreceptor degeneration, indicating a neuroprotective role for Bendless-PINK1-Park mediated Marf degradation. Based on our observations, we propose that certain forms of mitochondrial stress activate Bendless-PINK1-Park to limit mitochondrial fusion, which is a cell-protective response.

Development of S-aryl dithiocarbamate derived novel antiproliferative compound exhibiting tubulin bundling.

Cancer is a leading cause of human death, and there is a need to identify efficient and novel chemical scaffolds which could provide flexibility to cancer chemotherapeutics. This work introduces S-aryl dithiocarbamates belonging to a versatile group of organo-sulfur containing compounds as a hitherto unexplored class of effective anticancer drugs with promising pharmacophore properties. We synthesized a series of N-Boc piperazine containing S-aryl dithiocarbamates and identified compound 1 as a potent antiproliferative agent in lung, cervical, and breast cancer cell lines. Compound 1 exhibited best inhibitory activity against cervical cancer cells, HeLa with an IC50 of 0.432 ± 0.138 µM for 72 h, and lung cancer cells, A549 with an IC50 of 0.447 ± 0.051 µM for 72 h. We further demonstrate that HeLa cells treated with this compound result in G2/M phase cell cycle arrest, causing cell apoptosis due to the upregulation of the p53-p21 signaling pathway. Importantly, cells treated with compound 1 showed a novel tubulin bundling phenotype in fluorescence microscopy, which is a characteristic of microtubule-stabilizing anticancer drugs like paclitaxel. Interestingly, molecular docking analysis revealed reasonable binding of compound 1 in the taxol-binding pocket of ß-tubulin, making it a promising candidate for microtubule stabilization based anticancer drug discovery.

Multicomponent Synthesis of Biologically Relevant S-Aryl Dithiocarbamates Using Diaryliodonium Salts.

A transition-metal-free one-pot three-component annulation between diaryliodonium triflates, cyclic and acyclic aliphatic amines, and carbon disulfide providing a convenient and efficient access to biologically relevant S-aryl dithiocarbamates is developed. The reaction does not require metal, base, or any other additive and operates under mild and ambient conditions. This methodology is robust, scalable, and exhibits a broad substrate scope. The in silico analysis revealed that the majority of the compounds have a drug-likeness and good ADMET characteristics.

Centrosome dysfunction in human diseases.

Centrosomes are the major microtubule organizing centers in a large number of animal cells. They are involved in diverse cellular functions like cell division, migration, sensing and motility. Despite being identified more than 100 years ago, they did not receive much attention until recent discoveries suggesting their association with human diseases. Centrosome-related defects have been observed in several human diseases including cancers, brain disorders and ciliopathies. Researchers in the field are trying to understand the relationship between centrosomes and these diseases. Accordingly, this review provides an overview of the current knowledge regarding the role of centrosomes during ciliogenesis and neural stem cell division. The review primarily focuses on the impairment of centrosome number, organization and functioning leading to a wide range of human diseases. Finally, we discuss the scope of targeting centrosomes for therapeutic purposes.

Flip-flop Mediated Conditional Gene Inactivation in Drosophila.

Mosaic analysis in Drosophila, an important tool to assess cellular phenotypes of mutants in an otherwise heterozygous background, relies on mitosis. Hence, it cannot be used to inactivate gene function in mitotically inactive, terminally differentiated cells such as neurons. To address this issue, we developed "Flip-flop", a novel, Flippase-dependent in vivo cassette-inversion method that functions independent of mitosis, and therefore can be used for gene inactivation in both mitotic as well as postmitotic cells. This method allows tagging protein-coding genes with EGFP and generates mutant cells that are marked with mCherry upon cassette inversion. Here, we describe protocols for generation and validation of fly lines that can be used for conditional gene inactivation in mitotic as well as post-mitotic cells. We provide typical examples of Flip-flop mediated mosaic analysis in SNF4Aγ and Trim9. Use of Flip-flop mediated functional analysis will permit a detailed investigation of the role of genes previously recalcitrant to mosaic analysis.

Stereotyped terminal axon branching of leg motor neurons mediated by IgSF proteins DIP-α and Dpr10.

For animals to perform coordinated movements requires the precise organization of neural circuits controlling motor function. Motor neurons (MNs), key components of these circuits, project their axons from the central nervous system and form precise terminal branching patterns at specific muscles. Focusing on the Drosophila leg neuromuscular system, we show that the stereotyped terminal branching of a subset of MNs is mediated by interacting transmembrane Ig superfamily proteins DIP-α and Dpr10, present in MNs and target muscles, respectively. The DIP-α/Dpr10 interaction is needed only after MN axons reach the vicinity of their muscle targets. Live imaging suggests that precise terminal branching patterns are gradually established by DIP-α/Dpr10-dependent interactions between fine axon filopodia and developing muscles. Further, different leg MNs depend on the DIP-α and Dpr10 interaction to varying degrees that correlate with the morphological complexity of the MNs and their muscle targets.

Transsynaptic interactions between IgSF proteins DIP-α and Dpr10 are required for motor neuron targeting specificity.

The Drosophila larval neuromuscular system provides an ideal context in which to study synaptic partner choice, because it contains a small number of pre- and postsynaptic cells connected in an invariant pattern. The discovery of interactions between two subfamilies of IgSF cell surface proteins, the Dprs and the DIPs, provided new candidates for cellular labels controlling synaptic specificity. Here we show that DIP-α is expressed by two identified motor neurons, while its binding partner Dpr10 is expressed by postsynaptic muscle targets. Removal of either DIP-α or Dpr10 results in loss of specific axonal branches and NMJs formed by one motor neuron, MNISN-1s, while other branches of the MNISN-1s axon develop normally. The temporal and spatial expression pattern of dpr10 correlates with muscle innervation by MNISN-1s during embryonic development. We propose a model whereby DIP-α and Dpr10 on opposing synaptic partners interact with each other to generate proper motor neuron connectivity.

Neuron-Subtype-Specific Expression, Interaction Affinities, and Specificity Determinants of DIP/Dpr Cell Recognition Proteins.

Binding between DIP and Dpr neuronal recognition proteins has been proposed to regulate synaptic connections between lamina and medulla neurons in the Drosophila visual system. Each lamina neuron was previously shown to express many Dprs. Here, we demonstrate, by contrast, that their synaptic partners typically express one or two DIPs, with binding specificities matched to the lamina neuron-expressed Dprs. A deeper understanding of the molecular logic of DIP/Dpr interaction requires quantitative studies on the properties of these proteins. We thus generated a quantitative affinity-based DIP/Dpr interactome for all DIP/Dpr protein family members. This revealed a broad range of affinities and identified homophilic binding for some DIPs and some Dprs. These data, along with full-length ectodomain DIP/Dpr and DIP/DIP crystal structures, led to the identification of molecular determinants of DIP/Dpr specificity. This structural knowledge, along with a comprehensive set of quantitative binding affinities, provides new tools for functional studies in vivo.

Ari-1 Regulates Myonuclear Organization Together with Parkin and Is Associated with Aortic Aneurysms.

Nuclei are actively positioned and anchored to the cytoskeleton via the LINC (Linker of Nucleoskeleton and Cytoskeleton) complex. We identified mutations in the Parkin-like E3 ubiquitin ligase Ariadne-1 (Ari-1) that affect the localization and distribution of LINC complex members in Drosophila. ari-1 mutants exhibit nuclear clustering and morphology defects in larval muscles. We show that Ari-1 mono-ubiquitinates the core LINC complex member Koi. Surprisingly, we discovered functional redundancy between Parkin and Ari-1: increasing Parkin expression rescues ari-1 mutant phenotypes and vice versa. We further show that rare variants in the human homolog of ari-1 (ARIH1) are associated with thoracic aortic aneurysms and dissections, conditions resulting from smooth muscle cell (SMC) dysfunction. Human ARIH1 rescues fly ari-1 mutant phenotypes, whereas human variants found in patients fail to do so. In addition, SMCs obtained from patients display aberrant nuclear morphology. Hence, ARIH1 is critical in anchoring myonuclei to the cytoskeleton.

A cell cycle-independent, conditional gene inactivation strategy for differentially tagging wild-type and mutant cells.

Here, we describe a novel method based on intronic MiMIC insertions described in Nagarkar-Jaiswal et al. (2015) to perform conditional gene inactivation in Drosophila. Mosaic analysis in Drosophila cannot be easily performed in post-mitotic cells. We therefore, therefore, developed Flip-Flop, a flippase-dependent in vivo cassette-inversion method that marks wild-type cells with the endogenous EGFP-tagged protein, whereas mutant cells are marked with mCherry upon inversion. We document the ease and usefulness of this strategy in differential tagging of wild-type and mutant cells in mosaics. We use this approach to phenotypically characterize the loss of SNF4Aγ, encoding the γ subunit of the AMP Kinase complex. The Flip-Flop method is efficient and reliable, and permits conditional gene inactivation based on both spatial and temporal cues, in a cell cycle-, and developmental stage-independent fashion, creating a platform for systematic screens of gene function in developing and adult flies with unprecedented detail.

Loss of Nardilysin, a Mitochondrial Co-chaperone for α-Ketoglutarate Dehydrogenase, Promotes mTORC1 Activation and Neurodegeneration.

We previously identified mutations in Nardilysin (dNrd1) in a forward genetic screen designed to isolate genes whose loss causes neurodegeneration in Drosophila photoreceptor neurons. Here we show that NRD1 is localized to mitochondria, where it recruits mitochondrial chaperones and assists in the folding of α-ketoglutarate dehydrogenase (OGDH), a rate-limiting enzyme in the Krebs cycle. Loss of Nrd1 or Ogdh leads to an increase in α-ketoglutarate, a substrate for OGDH, which in turn leads to mTORC1 activation and a subsequent reduction in autophagy. Inhibition of mTOR activity by rapamycin or partially restoring autophagy delays neurodegeneration in dNrd1 mutant flies. In summary, this study reveals a novel role for NRD1 as a mitochondrial co-chaperone for OGDH and provides a mechanistic link between mitochondrial metabolic dysfunction, mTORC1 signaling, and impaired autophagy in neurodegeneration.

The E3 ligase Ubr3 regulates Usher syndrome and MYH9 disorder proteins in the auditory organs of Drosophila and mammals.

Myosins play essential roles in the development and function of auditory organs and multiple myosin genes are associated with hereditary forms of deafness. Using a forward genetic screen in Drosophila, we identified an E3 ligase, Ubr3, as an essential gene for auditory organ development. Ubr3 negatively regulates the mono-ubiquitination of non-muscle Myosin II, a protein associated with hearing loss in humans. The mono-ubiquitination of Myosin II promotes its physical interaction with Myosin VIIa, a protein responsible for Usher syndrome type IB. We show that ubr3 mutants phenocopy pathogenic variants of Myosin II and that Ubr3 interacts genetically and physically with three Usher syndrome proteins. The interactions between Myosin VIIa and Myosin IIa are conserved in the mammalian cochlea and in human retinal pigment epithelium cells. Our work reveals a novel mechanism that regulates protein complexes affected in two forms of syndromic deafness and suggests a molecular function for Myosin IIa in auditory organs.

Single-cell analysis of transcription kinetics across the cell cycle.

Transcription is a highly stochastic process. To infer transcription kinetics for a gene-of-interest, researchers commonly compare the distribution of mRNA copy-number to the prediction of a theoretical model. However, the reliability of this procedure is limited because the measured mRNA numbers represent integration over the mRNA lifetime, contribution from multiple gene copies, and mixing of cells from different cell-cycle phases. We address these limitations by simultaneously quantifying nascent and mature mRNA in individual cells, and incorporating cell-cycle effects in the analysis of mRNA statistics. We demonstrate our approach on Oct4 and Nanog in mouse embryonic stem cells. Both genes follow similar two-state kinetics. However, Nanog exhibits slower ON/OFF switching, resulting in increased cell-to-cell variability in mRNA levels. Early in the cell cycle, the two copies of each gene exhibit independent activity. After gene replication, the probability of each gene copy to be active diminishes, resulting in dosage compensation.

Ig Superfamily Ligand and Receptor Pairs Expressed in Synaptic Partners in Drosophila.

Information processing relies on precise patterns of synapses between neurons. The cellular recognition mechanisms regulating this specificity are poorly understood. In the medulla of the Drosophila visual system, different neurons form synaptic connections in different layers. Here, we sought to identify candidate cell recognition molecules underlying this specificity. Using RNA sequencing (RNA-seq), we show that neurons with different synaptic specificities express unique combinations of mRNAs encoding hundreds of cell surface and secreted proteins. Using RNA-seq and protein tagging, we demonstrate that 21 paralogs of the Dpr family, a subclass of immunoglobulin (Ig)-domain containing proteins, are expressed in unique combinations in homologous neurons with different layer-specific synaptic connections. Dpr interacting proteins (DIPs), comprising nine paralogs of another subclass of Ig-containing proteins, are expressed in a complementary layer-specific fashion in a subset of synaptic partners. We propose that pairs of Dpr/DIP paralogs contribute to layer-specific patterns of synaptic connectivity.

Control of Synaptic Connectivity by a Network of Drosophila IgSF Cell Surface Proteins.

We have defined a network of interacting Drosophila cell surface proteins in which a 21-member IgSF subfamily, the Dprs, binds to a nine-member subfamily, the DIPs. The structural basis of the Dpr-DIP interaction code appears to be dictated by shape complementarity within the Dpr-DIP binding interface. Each of the six dpr and DIP genes examined here is expressed by a unique subset of larval and pupal neurons. In the neuromuscular system, interactions between Dpr11 and DIP-γ affect presynaptic terminal development, trophic factor responses, and neurotransmission. In the visual system, dpr11 is selectively expressed by R7 photoreceptors that use Rh4 opsin (yR7s). Their primary synaptic targets, Dm8 amacrine neurons, express DIP-γ. In dpr11 or DIP-γ mutants, yR7 terminals extend beyond their normal termination zones in layer M6 of the medulla. DIP-γ is also required for Dm8 survival or differentiation. Our findings suggest that Dpr-DIP interactions are important determinants of synaptic connectivity.

A genetic toolkit for tagging intronic MiMIC containing genes.

Previously, we described a large collection of Minos-Mediated Integration Cassettes (MiMICs) that contain two phiC31 recombinase target sites and allow the generation of a new exon that encodes a protein tag when the MiMIC is inserted in a codon intron (Nagarkar-Jaiswal et al., 2015). These modified genes permit numerous applications including assessment of protein expression pattern, identification of protein interaction partners by immunoprecipitation followed by mass spec, and reversible removal of the tagged protein in any tissue. At present, these conversions remain time and labor-intensive as they require embryos to be injected with plasmid DNA containing the exon tag. In this study, we describe a simple and reliable genetic strategy to tag genes/proteins that contain MiMIC insertions using an integrated exon encoding GFP flanked by FRT sequences. We document the efficiency and tag 60 mostly uncharacterized genes.

A library of MiMICs allows tagging of genes and reversible, spatial and temporal knockdown of proteins in Drosophila.

Here, we document a collection of ∼7434 MiMIC (Minos Mediated Integration Cassette) insertions of which 2854 are inserted in coding introns. They allowed us to create a library of 400 GFP-tagged genes. We show that 72% of internally tagged proteins are functional, and that more than 90% can be imaged in unfixed tissues. Moreover, the tagged mRNAs can be knocked down by RNAi against GFP (iGFPi), and the tagged proteins can be efficiently knocked down by deGradFP technology. The phenotypes associated with RNA and protein knockdown typically correspond to severe loss of function or null mutant phenotypes. Finally, we demonstrate reversible, spatial, and temporal knockdown of tagged proteins in larvae and adult flies. This new strategy and collection of strains allows unprecedented in vivo manipulations in flies for many genes. These strategies will likely extend to vertebrates.

A voltage-gated calcium channel regulates lysosomal fusion with endosomes and autophagosomes and is required for neuronal homeostasis.

Autophagy helps deliver sequestered intracellular cargo to lysosomes for proteolytic degradation and thereby maintains cellular homeostasis by preventing accumulation of toxic substances in cells. In a forward mosaic screen in Drosophila designed to identify genes required for neuronal function and maintenance, we identified multiple cacophony (cac) mutant alleles. They exhibit an age-dependent accumulation of autophagic vacuoles (AVs) in photoreceptor terminals and eventually a degeneration of the terminals and surrounding glia. cac encodes an α1 subunit of a Drosophila voltage-gated calcium channel (VGCC) that is required for synaptic vesicle fusion with the plasma membrane and neurotransmitter release. Here, we show that cac mutant photoreceptor terminals accumulate AV-lysosomal fusion intermediates, suggesting that Cac is necessary for the fusion of AVs with lysosomes, a poorly defined process. Loss of another subunit of the VGCC, α2δ or straightjacket (stj), causes phenotypes very similar to those caused by the loss of cac, indicating that the VGCC is required for AV-lysosomal fusion. The role of VGCC in AV-lysosomal fusion is evolutionarily conserved, as the loss of the mouse homologues, Cacna1a and Cacna2d2, also leads to autophagic defects in mice. Moreover, we find that CACNA1A is localized to the lysosomes and that loss of lysosomal Cacna1a in cerebellar cultured neurons leads to a failure of lysosomes to fuse with endosomes and autophagosomes. Finally, we show that the lysosomal CACNA1A but not the plasma-membrane resident CACNA1A is required for lysosomal fusion. In summary, we present a model in which the VGCC plays a role in autophagy by regulating the fusion of AVs with lysosomes through its calcium channel activity and hence functions in maintaining neuronal homeostasis.

The cohesin subunit Rad21 is required for synaptonemal complex maintenance, but not sister chromatid cohesion, during Drosophila female meiosis.

Replicated sister chromatids are held in close association from the time of their synthesis until their separation during the next mitosis. This association is mediated by the ring-shaped cohesin complex that appears to embrace the sister chromatids. Upon proteolytic cleavage of the α-kleisin cohesin subunit at the metaphase-to-anaphase transition by separase, sister chromatids are separated and segregated onto the daughter nuclei. The more complex segregation of chromosomes during meiosis is thought to depend on the replacement of the mitotic α-kleisin cohesin subunit Rad21/Scc1/Mcd1 by the meiotic paralog Rec8. In Drosophila, however, no clear Rec8 homolog has been identified so far. Therefore, we have analyzed the role of the mitotic Drosophila α-kleisin Rad21 during female meiosis. Inactivation of an engineered Rad21 variant by premature, ectopic cleavage during oogenesis results not only in loss of cohesin from meiotic chromatin, but also in precocious disassembly of the synaptonemal complex (SC). We demonstrate that the lateral SC component C(2)M can interact directly with Rad21, potentially explaining why Rad21 is required for SC maintenance. Intriguingly, the experimentally induced premature Rad21 elimination, as well as the expression of a Rad21 variant with destroyed separase consensus cleavage sites, do not interfere with chromosome segregation during meiosis, while successful mitotic divisions are completely prevented. Thus, chromatid cohesion during female meiosis does not depend on Rad21-containing cohesin.