The Selective Autophagy Receptor p62 Forms a Flexible Filamentous Helical Scaffold

Published version also available at http://dx.doi.org/10.1016/j.celrep.2015.03.062The scaffold protein p62/SQSTM1 is involved in protein turnover and signaling and is commonly found in dense protein bodies in eukaryotic cells. In autophagy, p62 acts as a selective autophagy receptor that recognizes and shuttles ubiquitinated proteins to the autophagosome for degradation. The structural organization of p62 in cellular bodies and the interplay of these assemblies with ubiquitin and the autophagic marker LC3 remain to be elucidated. Here, we present a cryo-EM structural analysis of p62. Together with structures of assemblies from the PB1 domain, we show that p62 is organized in flexible polymers with the PB1 domain constituting a helical scaffold. Filamentous p62 is capable of binding LC3 and addition of long ubiquitin chains induces disassembly and shortening of filaments. These studies explain how p62 assemblies provide a large molecular scaffold for the nascent autophagosome and reveal how they can bind ubiquitinated cargo

ISWI is a RanGTP-dependent MAP required for chromosome segregation

Chromatin-remodeling factor ISWI is a microtubule-associated protein that contributes to chromosome segregation by stabilizing microtubules during anaphase

MURF2B, a Novel LC3-Binding Protein, Participates with MURF2A in the Switch between Autophagy and Ubiquitin Proteasome System during Differentiation of C2C12 Muscle Cells.

International audienceThe ubiquitin proteasome system and macroautophagy are proteolytic pathways essential in the maintenance of cellular homeostasis during differentiation and remodelling of skeletal muscle. In both pathways, proteins to be degraded are tagged with polyubiquitin. In skeletal muscles, the MURF2 proteins display E3 ubiquitin ligase structure suggesting that they may covalently attach ubiquitin polypeptides to still unknown target proteins. So far only MURF2A isoforms were studied and shown to interact with p62/SQSTM1, a protein implicated in macroautophagic and ubiquitin proteasome system degradations. Here, we analyzed the MURF2B and MURF2A proteins and show that the ratio of the isoforms changes during differentiation of muscle C2C12 cells and that the shift of the isoforms expression follows the sequential activation of autophagic or proteasomal degradation. We also show that MURF2B has a functional domain needed for its interaction with LC3, a protein needed for autophagic vesicles formation. Using specific MURF2 RNAi cells we observed that MURF2A and MURF2B are both needed for the formation of autophagosomes and that in the absence of MURF2B, the cells expressing MURF2A display an activated ubiquitin proteasome system implicated in the degradation of p62/SQSTM1 by UPS. Altogether, our results indicate that MURF2A and MURF2B proteins could participate in the molecular switch between the two ubiquitin degradative pathways

CHD4 Is a RanGTP-Dependent MAP that Stabilizes Microtubules and Regulates Bipolar Spindle Formation

Background Production of the GTP-bound form of the Ran GTPase (RanGTP) around chromosomes induces spindle assembly by activating nuclear localization signal (NLS)-containing proteins. Several NLS proteins have been identified as spindle assembly factors, but the complexity of the process led us to search for additional proteins with distinct roles in spindle assembly. Results We identify a chromatin-remodeling ATPase, CHD4, as a RanGTP-dependent microtubule (MT)-associated protein (MAP). MT binding occurs via the region containing an NLS and chromatin-binding domains. In Xenopus egg extracts and cultured cells, CHD4 largely dissociates from mitotic chromosomes and partially localizes to the spindle. Immunodepletion of CHD4 from egg extracts significantly reduces the quantity of MTs produced around chromatin and prevents spindle assembly. CHD4 RNAi in both HeLa and Drosophila S2 cells induces defects in spindle assembly and chromosome alignment in early mitosis, leading to chromosome missegregation. Further analysis in egg extracts and in HeLa cells reveals that CHD4 is a RanGTP-dependent MT stabilizer. Moreover, the CHD4-containing NuRD complex promotes organization of MTs into bipolar spindles in egg extracts. Importantly, this function of CHD4 is independent of chromatin remodeling. Conclusions Our results uncover a new role for CHD4 as a MAP required for MT stabilization and involved in generating spindle bipolarity. © 2013 Elsevier Ltd

During myogenic differentiation, various MURF2 complexes are formed.

<p>(A) MURF2A and MURF2B co-localize in C2C12 myoblasts. C2C12 cells were transfected with mCherry-MURF2A. After 24h, endogenous MURF2B was detected using MURF2B antibody (green) and fluorescent MURF2A was observed directly (red). The magnified region reveals that MURF2A and MURF2B are in close contact (arrows). Scale Bars: 10 µm. (B) MURF2A and MURF2B are complexed with LC3 and p62. C2C12 cells transfected with GFP-LC3 or GFP-p62 were kept undifferentiated (Diff. 0h) or differentiated for 24h (Diff. 24h) in absence (-) or in presence (+) of 200 nM Baf for 4 h before cell lysis in RIPA buffer. Western blots were performed with precipitated proteins obtained using magnetic beads (Bio-Adembeads pAG) incubated with 300 µg extract complemented (+) or not (-) with anti-GFP antibody. Western blots (WB) were probed with the indicated antibodies. Asterisks indicate unspecific signals.</p

MURF2A activates the 20S proteasome and is implicated in p62 degradation.

<p>(A) Undifferentiated stable C2C12 and RNAi cell lines expressing the mCherry-GFP-LC3 protein were grown in 10% FCS and observed directly by confocal microscopy. In magnified regions, the yellow autophagosomes are indicated by empty arrowheads. (B) The same cell lines were kept in HBSS medium for 45 min and observed directly by confocal microscopy. Magnified regions show in C2C12 cells yellow autophagosomes (empty arrowheads) and red autolysosomes (arrows). In RNAi cells unconventional autophagic structures are indicated by arrowheads. Scale Bar: 10 µm.</p

MURF2 expression during differentiation of C2C12 cells.

<p>(A) Schematic structure of the MURF2 isoforms of skeletal muscle cells and locations of the epitopes recognized by various antibodies (names are framed). (B) Same amounts of C2C12 lysates obtained at various time of differentiation (Diff.) were used for Western blots analyses and probed with the indicated antibodies.</p

(A) Representative immunoblots of C2C12 lysates probed with poly ubiquitin, the α5 subunit of the 20S proteasome, myogenin, Troponin T and GAPDH antibodies.

<p>Lysates were obtained at various times of differentiation (Diff.). (B) Activity of the 20S proteasome in C2C12 cells. Proteolytic 20S proteasome chymotrypsin-like activity of the indicated undifferentiated (0h) and differentiated (48h) C2C12 cells was determined measuring the amidomethylcoumarin (AMC) generated as the cleavage product of the fluorogenic substrat succinyl-leucine-leucine-valine-tyrosine-7 amido-4-methyl-coumarin. Differences were evaluated by the Kruskal-Wallis test. Medians and quartiles are shown in the histograms. Compared to C2C12 myoblasts (Diff. 0h), myotubes (Diff. 48h) showed significant differences (*, P<0.05). (C) Activity of the 20S proteasome in various RNAi cell lines. The proteolytic 20S proteasome chymotrypsin-like activity was determined for undifferentiated (0h) and differentiated (48h) cells as previously mentioned for C2C12 cells. Compared to C2C12 and RNAi MURF2A, RNAi MURF2B cells showed significant increase of their 20S proteasome activity (*, P<0.05). (D) Effect of UPS and autophagic inhibition on p62 expression in C2C12 and MURF2 RNAi cells. After 24h of differentiation in 1% horse serum medium (1% HS), cells were shifted to fresh differentiation medium (1% HS) or treated for 2h 30min with fresh differentiation medium complemented with 50 µM MG132 and 200 nM Baf (1% HS+MG132+Baf) before lysis. Western blots were probed with anti p62, anti mono and poly ubiquitin and anti GAPDH antibodies. (E) p62 is degraded by UPS in differentiated C2C12 cells. C2C12 cells were transfected with vectors encoding GFP or GFP-p62 under the CMV promoter. 24h after transfection, cells were split into two groups, induced to differentiate for 24h, then shifted to fresh differentiation medium containing (+) or not (-) 10 µM MG132 for 12h. Extracts were used for Western blot analyses and probed with the indicated antibodies. Rabbit anti mono and poly ubiquitin antibody was used.</p