Nudel is crucial for the WAVE complex assembly in vivo by selectively promoting subcomplex stability and formation through direct interactions

The WAVE regulatory complex (WRC), consisting of WAVE, Sra, Nap, Abi, and HSPC300, activates the Arp2/3 complex to control branched actin polymerization in response to Rac activation. How the WRC is assembled in vivo is not clear. Here we show that Nudel, a protein critical for lamellipodia formation, dramatically stabilized the Sra1-Nap1-Abi1 complex against degradation in cells through a dynamic binding to Sra1, whereas its physical interaction with HSPC300 protected free HSPC300 from the proteasome-mediated degradation and stimulated the HSPC300-WAVE2 complex formation. By contrast, Nudel showed little or no interactions with the Sra1-Nap1-Abi1-WAVE2 and the Sra1-Nap1-Abi1-HSPC300 complexes as well as the mature WRC. Depletion of Nudel by RNAi led to general subunit degradation and markedly attenuated the levels of mature WRC. It also abolished the WRC-dependent actin polymerization in vitro and the Rac1-induced lamellipodial actin network formation during cell spreading. Therefore, Nudel is important for the early steps of the WRC assembly in vivo by antagonizing the instability of certain WRC subunits and subcomplexes.     

Ndel1 alters its conformation by sequestering cAMP-specific phosphodiesterase-4D3 (PDE4D3) in a manner that is dynamically regulated through Protein Kinase A (PKA)

The involvement of the Nuclear distribution element-like (Ndel1; Nudel) protein in the recruitment of the dynein complex is critical for neurodevelopment and potentially important for neuronal disease states. The PDE4 family of phosphodiesterases specifically degrades cAMP, an important second messenger implicated in learning and memory functions. Here we show for the first time that Ndel1 can interact directly with PDE4 family members and that the interaction of Ndel1 with the PDE4D3 isoform is uniquely disrupted by elevation of intracellular cAMP levels. While all long PDE4 isoforms are subject to stimulatory PKA phosphorylation within their conserved regulatory UCR1 domain, specificity for release of PDE4D3 is conferred due to the PKA-dependent phosphorylation of Ser13 within the isoform-specific, unique amino-terminal domain of PDE4D3. Scanning peptide array analyses identify a common region on Ndel1 for PDE4 binding and an additional region that is unique to PDE4D3. The common site lies within the stutter region that links the second coiled-coil region to the unstable third coiled-coil regions of Ndel1. The additional binding region unique to PDE4D3 penetrates into the start of the third coiled-coil region that can undergo tail-to-tail interactions between Ndel1 dimers to form a 4 helix bundle. We demonstrate Ndel1 self-interaction in living cells using a BRET approach with luciferase- and GFP-tagged forms of Ndel1. BRET assessed Ndel1–Ndel1 self-interaction is amplified through the binding of PDE4 isoforms. For PDE4D3 this effect is ablated upon elevation of intracellular cAMP due to PKA-mediated phosphorylation at Ser13, while the potentiating effects of PDE4B1 and PDE4D5 are resistant to cAMP elevation. PDE4D long isoforms and Ndel1 show a similar sub-cellular distribution in hippocampus and cortex and locate to post-synaptic densities. We show that Ndel1 sequesters EPAC, but not PKA, in order to form a cAMP signalling complex. We propose that a key function of the Ndel1 signalling scaffold is to signal through cAMP by sequestering EPAC, whose activity may thus be specifically regulated by sequestered PDE4 that also stabilizes Ndel1–Ndel1 self-interaction. In the case of PDE4D3, its association with Ndel1 is dynamically regulated by PKA input through its ability to phosphorylate Ser13 in the unique N-terminal region of this isoform, triggering the specific release of PDE4D3 from Ndel1 when cAMP levels are elevated. We propose that Ser13 may act as a redistribution trigger in PDE4D3, allowing it to dynamically re-shape cAMP gradients in distinct intracellular locales upon its phosphorylation by PKA.     

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 p150^Glued 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.     

Identification of a novel dynein binding domain in nudel essential for spindle pole organization in Xenopus egg extract

The nuclear distribution protein E (NudE) and nuclear distribution protein E-like (Nudel or Ndel1) interact with both lissencephaly 1 (Lis1) and dynein. These interactions are thought to be essential for dynein function. Previous studies have shown that the highly conserved N terminus of NudE/Nudel directly binds to Lis1, and such binding is critical for dynein activity. By contrast, although the C terminus of NudE/Nudel was reported to bind to dynein, the functional significance of this binding has remained unclear. Using the sperm-mediated spindle assembly assay in Xenopus egg extracts and extensive mutagenesis studies, we have identified a highly conserved dynein binding domain within the first 80 amino acids of Nudel. We further demonstrate that the dynein intermediate chain in the dynein complex is directly involved in this interaction. Importantly, we show that both the dynein and Lis1 binding domains of Nudel are required for spindle pole organization. Finally, we report that spindle defects caused by immuno-depletion of Nudel could be rescued by a 1-fold increase of Lis1 concentration in Xenopus egg extracts. This suggests that an important function of the N terminus of Nudel is to facilitate the interaction between Lis1 and dynein during spindle assembly. Together, our findings open up new avenues to further decipher the mechanism of dynein regulation by Nudel and Lis1.