Yidong Shen

Human Nudel and NudE as Regulators of Cytoplasmic Dynein in Poleward Protein Transport along the Mitotic Spindle

ABSTRACT Emerging evidence supports the idea that a signaling pathway containing orthologs of at least mammalian NudE and Nudel, Lis1, and cytoplasmic dynein is conserved for eukaryotic nuclear migration. In mammals, this pathway has profound impact on neuronal migration during development of the central nervous system. Lis1 and dynein are also involved in other cellular functions, such as mitosis. Here we show that Nudel also participates in a subset of dynein function in M phase. Nudel was specifically phosphorylated in M phase in its serine/threonine phosphorylation motifs, probably by Cdc2 and also Erk1 and -2. A fraction of Nudel bound to centrosomes strongly in interphase and localized to mitotic spindles in early M phase. By using mutants incapable of or simulating phosphorylation, we confirmed that phosphorylation of Nudel regulated the cell-cycle-dependent distribution, possibly by increasing its dissociation rate at the microtubule-organizing center. Moreover, phosphorylated Nudel or the phosphorylation-mimicking mutant bound Lis1 more efficiently. We further demonstrated that a Nudel mutant incapable of binding to Lis1 impaired the poleward movement of dynein and hence the dynein-mediated transport of kinetochore proteins to spindle poles along microtubules, a process contributing to inactivation of the spindle checkpoint in mitosis. These results point to the importance of Nudel-Lis1 interaction for the dynein activity in M phase and to a possible role of Nudel phosphorylation as facilitating such interaction. In addition, comparative studies suggest that NudE is also functionally related to its paralog, Nudel.

miR-129-3p controls cilia assembly by regulating CP110 and actin dynamics

Ciliogenesis requires the removal of CP110 from the mother centriole; actin dynamics also influence ciliation, at least partly by affecting the centrosomal accumulation of ciliogenic membrane vesicles. How these distinct processes are properly regulated remains unknown. Here we show that miR-129-3p, a microRNA conserved in vertebrates, controlled cilia biogenesis in cultured cells by concomitantly downregulating CP110 and repressing branched F-actin formation. Blocking miR-129-3p inhibited serum-starvation-induced ciliogenesis, whereas its overexpression potently induced ciliation in proliferating cells and also promoted cilia elongation. Gene expression analysis further identified ARP2, TOCA1, ABLIM1 and ABLIM3 as its targets in ciliation-related actin dynamics. Moreover, miR-129-3p inhibition in zebrafish embryos suppressed ciliation in Kupffer’s vesicle and the pronephros, and induced developmental abnormalities including a curved body, pericardial oedema and defective left–right asymmetry. Therefore, our results reveal a mechanism that orchestrates both the centriole-to-basal body transition and subsequent cilia assembly through microRNA-mediated post-transcriptional regulation.

A Steroid Receptor–MicroRNA Switch Regulates Life Span in Response to Signals from the Gonad

Gonads and Life Span Animals develop through successive life stages and have life spans that are determined by their genes and the environment. Yet how the molecular circuitry underlying life-stage structure relates to longevity is poorly understood. Shen et al. (p. 1472) now show that components of a steroid receptor-microRNA switch used for Caenorhabditis elegans developmental timing is co-opted to regulate adult longevity in response to signals from the reproductive system. Thus, the gonad can be coupled to metazoan life span via a developmental clock. The mechanism by which removal of signals from the reproductive organs promotes longevity in nematodes is elucidated. Although the gonad primarily functions in procreation, it also affects animal life span. Here, we show that removal of the Caenorhabditis elegans germ line triggers a switch in the regulatory state of the organism to promote longevity, co-opting components involved in larval developmental timing circuits. These components include the DAF-12 steroid receptor, which is involved in the larval stage two–to–stage three (L2-L3) transition and up-regulates members of the let-7 microRNA (miRNA) family. The miRNAs target an early larval nuclear factor lin-14 and akt-1/kinase, thereby stimulating DAF-16/FOXO signaling to extend life. Our studies suggest that metazoan life span is coupled to the gonad through elements of a developmental timer.

Nudel Binds Cdc42GAP to Modulate Cdc42 Activity at the Leading Edge of Migrating Cells

Cdc42GAP promotes inactivation of Cdc42, a small GTPase whose activation at the leading edge by guanine nucleotide exchange factors is critical for cell migration. How Cdc42GAP is regulated to ensure proper levels of active Cdc42 is poorly understood. Here we show that Nudel, a cytoplasmic dynein regulator, competes with Cdc42 for binding Cdc42GAP. Consequently, Nudel can inhibit Cdc42GAP-mediated inactivation of Cdc42 in a dose-dependent manner. Both Nudel and Cdc42GAP exhibit leading-edge localization in migrating cells. The localization of Nudel requires its phosphorylation by Erk1/2. Depleting Nudel by RNAi or overexpression of a nonphosphorylatable mutant abolishes Cdc42 activation and cell migration. Our data thus uncover Nudel as a regulator of Cdc42 during cell migration. Nudel facilitates cell migration by sequestering Cdc42GAP at the leading edge to stabilize active Cdc42 in response to extracellular stimuli. Excess active Cdc42 may in turn control its own activity by recruiting Cdc42GAP from Nudel.

A novel 3-hydroxysteroid dehydrogenase that regulates reproductive development and longevity

A multidisciplinary approach identifies novel biochemical activities involved in the synthesisof C. elegans bile acid-like steroids, which act as hormones that regulate sterol metabolism and longevity.

Dietary Restriction Induced Longevity Is Mediated by Nuclear Receptor NHR-62 in Caenorhabditis elegans

Dietary restriction (DR) extends lifespan in a wide variety of species, yet the underlying mechanisms are not well understood. Here we show that the Caenorhabditis elegans HNF4α-related nuclear hormone receptor NHR-62 is required for metabolic and physiologic responses associated with DR-induced longevity. nhr-62 mediates the longevity of eat-2 mutants, a genetic mimetic of dietary restriction, and blunts the longevity response of DR induced by bacterial food dilution at low nutrient levels. Metabolic changes associated with DR, including decreased Oil Red O staining, decreased triglyceride levels, and increased autophagy are partly reversed by mutation of nhr-62. Additionally, the DR fatty acid profile is altered in nhr-62 mutants. Expression profiles reveal that several hundred genes induced by DR depend on the activity of NHR-62, including a putative lipase required for the DR response. This study provides critical evidence of nuclear hormone receptor regulation of the DR longevity response, suggesting hormonal and metabolic control of life span.

The NHR-8 Nuclear Receptor Regulates Cholesterol and Bile Acid Homeostasis in C. elegans

Hormone-gated nuclear receptors (NRs) are conserved transcriptional regulators of metabolism, reproduction, and homeostasis. Here we show that C. elegans NHR-8 NR, a homolog of vertebrate liver X and vitamin D receptors, regulates nematode cholesterol balance, fatty acid desaturation, apolipoprotein production, and bile acid metabolism. Loss of nhr-8 results in a deficiency in bile acid-like steroids, called the dafachronic acids, which regulate the related DAF-12/NR, thus controlling entry into the long-lived dauer stage through cholesterol availability. Cholesterol supplementation rescues various nhr-8 phenotypes, including developmental arrest, unsaturated fatty acid deficiency, reduced fertility, and shortened life span. Notably, nhr-8 also interacts with daf-16/FOXO to regulate steady-state cholesterol levels and is synthetically lethal in combination with insulin signaling mutants that promote unregulated growth. Our studies provide important insights into nuclear receptor control of cholesterol balance and metabolism and their impact on development, reproduction, and aging in the context of larger endocrine networks.

The Rieske oxygenase DAF-36 functions as a cholesterol 7-desaturase in steroidogenic pathways governing longevity

Bile acids are cholesterol‐derived signaling molecules that regulate mammalian metabolism through sterol‐sensing nuclear receptor transcription factors. In C. elegans, bile acid‐like steroids called dafachronic acids (DAs) control developmental timing and longevity by activating the nuclear receptor DAF‐12. However, little is known about the biosynthesis of these molecules. Here, we show that the DAF‐36/Rieske oxygenase works at the first committed step, converting cholesterol to 7‐dehydrocholesterol. Its elucidation as a cholesterol 7‐desaturase provides crucial biochemical evidence that such oxygenases are key steroidogenic enzymes. By controlling DA production, DAF‐36 regulates DAF‐12 activities for reproductive development and longevity and may illuminate related pathways in metazoans.

Nudel Promotes Axonal Lysosome Clearance and Endo‐lysosome Formation via Dynein‐Mediated Transport

Axonal transport is critical for neuronal function and survival. Cytoplasmic dynein and its accessory complex dynactin form a microtubule minus end‐directed motor in charge of retrograde transport. In this study, we show that Nudel, a dynein regulator, was highly expressed in dorsal root ganglion (DRG) neurons. Microinjection of anti‐Nudel antibody into cultured DRG neurons abolished retrograde transport of membranous organelles in the axon and led to dispersions of Golgi cisternae in the soma. As a result, lysosomes, which are normally enriched in the soma, moved persistently into and thus accumulated in axons. Endo‐lysosome formation was also markedly delayed. As anterograde motility of mitochondria was not inhibited, the antibody apparently did not abolish retrograde transport by destructing axonal microtubule tracks. Similar results were obtained by microinjecting N‐terminal Nudel, anti‐dynein antibody or a p150Glued mutant capable of abrogating the dynein–dynactin association. These results indicate a critical role of Nudel in dynein‐mediated axonal transport. Moreover, the effects of dynein on endolysosome formation and regional sequestration of lysosomes may contribute to defects in the endocytic pathway seen in neurons of patients or animals with malfunction of dynein.

Live Show of Rho GTPases in Cell Migration

Rho GTPases, including RhoA, Rac1 and Cdc42, are key regulators of cell migration in animals. In a recent issue of Nature, two papers present delicate studies on precise roles and spatiotemporal coordination of Rho GTPases in live cells, using either a photoactivatable Rac1 or specific biosensors, together with computational multiplexing method. In addition to implications in cell migration, analytical tools and methods used in these works can also be extended into other live cell studies on complex cellular/molecular events.