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

Rab27a targeting to melanosomes requires nucleotide exchange but not effector binding

Rab GTPases are important determinants of organelle identity and regulators of vesicular transport pathways. Consequently, each Rab occupies a highly specific subcellular localization. However, the precise mechanisms governing Rab targeting remain unclear. Guanine nucleotide exchange factors (GEFs), putative membrane-resident targeting factors and effector binding have all been implicated as critical regulators of Rab targeting. Here, we address these issues using Rab27a targeting to melanosomes as a model system. Rab27a regulates motility of lysosome-related organelles and secretory granules. Its effectors have been characterized extensively, and we have identified Rab3GEP as the non-redundant Rab27a GEF in melanocytes (Figueiredo AC et al. Rab3GEP is the non-redundant guanine nucleotide exchange factor for Rab27a in melanocytes. J Biol Chem 2008;283:23209-23216). Using Rab27a mutants that show impaired binding to representatives of all four Rab27a effector subgroups, we present evidence that effector binding is not essential for targeting of Rab27a to melanosomes. In contrast, we observed that knockdown of Rab3GEP resulted in mis-targeting of Rab27a, suggesting that Rab3GEP activity is required for correct targeting of Rab27a. However, the identification of Rab27a mutants that undergo efficient GDP/GTP exchange in the presence of Rab3GEP in vitro but are mis-targeted in a cellular context indicates that nucleotide loading is not the sole determinant of subcellular targeting of Rab27a. Our data support a model in which exchange activity, but not effector binding, represents one essential factor that contributes to membrane targeting of Rab proteins.publishersversionpublishe

Rab3GEP Is the Non-redundant Guanine Nucleotide Exchange Factor for Rab27a in Melanocytes *

Rab GTPases regulate discrete steps in vesicular transport pathways. Rabs require activation by specific guanine nucleotide exchange factors (GEFs) that stimulate the exchange of GDP for GTP. Rab27a controls motility and regulated exocytosis of secretory granules and related organelles. In melanocytes, Rab27a regulates peripheral transport of mature melanosomes by recruiting melanophilin and myosin Va. Here, we studied the activation of Rab27a in melanocytes. We identify Rab3GEP, previously isolated as a GEF for Rab3a, as the non-redundant Rab27a GEF. Similar to Rab27a-deficient ashen melanocytes, Rab3GEP-depleted cells show both clustering of melanosomes in the perinuclear area and loss of the Rab27a effector Mlph. Consistent with a role as an activator, levels of Rab27a-GTP are decreased in cells lacking Rab3GEP. Recombinant Rab3GEP exhibits guanine nucleotide exchange activity against Rab27a and Rab27b in vitro, in addition to its previously documented activity against Rab3. Our results indicate promiscuity in Rab GEF action and suggest that members of related but functionally distinct Rab subfamilies can be controlled by common activators

Melanin transferred to keratinocytes resides in non-degradative endocytic compartments

Funding: We thank the staff from the Unit of Imaging and Cytometry of the Instituto Gulbenkian de Ciência for assistance in microscopy and flow cytometry protocol adjustments, the CEDOC Microscopy Facility for assistance in image acquisition and analysis, Dot Bennett for the kind gift of cell lines, and Alistair Hume for the kind gift of anti-Rab5 antibody. This project was supported by Fundação para a Ciência e a Tecnologia (FCT), Portugal (PTDC/BIA-BCM/ 111735/2009, EXPL/BEX-BCM/0379/2013), MSC and HM were supported by FCT PhD studentships (SFRH/BD/65381/2009 and PD/BD/114118/2015, respectively), FJCP was supported by an FCT postdoctoral fellowship (SFRH/ BPD/70337/2010), and DCB was supported by the FCT Investigator Program (IF/00501/2014/CP1252/CT0001).Melanin transfer from melanocytes to keratinocytes and subsequent accumulation in the supra-nuclear region is a critical process in skin pigmentation and protection against ultraviolet radiation. We have previously proposed that the main mode of transfer between melanocytes and keratinocytes is through exo/endocytosis of the melanosome core, termed melanocore. In this study, we developed an in vitro uptake assay using melanocores secreted by melanocytes. We show that the uptake of melanocores, but not melanosomes, by keratinocytes is Protease-activated receptor (PAR)-2-dependent. Furthermore, we found that the silencing of the early endocytic regulator Rab5b, but not the late endocytic regulators Rab7a or Rab9a, significantly impairs melanocore uptake by keratinocytes. Following uptake, we observed that melanin accumulates in compartments that are positive for both early and late endocytic markers. We found that melanin does not localize to either highly degradative or acidic organelles, as assessed by LysoTracker and DQ-BSA staining, despite the abundance of these types of organelles within keratinocytes. Therefore, we propose that melanocore uptake leads to storage of melanin within keratinocytes in hybrid endocytic compartments that are not highly acidic or degradative. By avoiding lysosomal degradation, these specialized endosomes may allow melanin to persist within keratinocytes for long periods.publishersversionpublishe

Biophysical basis of filamentous phage tactoid-mediated antibiotic tolerance in P. aeruginosa

Inoviruses are filamentous phages infecting numerous prokaryotic phyla. Inoviruses can self-assemble into mesoscale structures with liquid-crystalline order, termed tactoids, which protect bacterial cells in Pseudomonas aeruginosa biofilms from antibiotics. Here, we investigate the structural, biophysical, and protective properties of tactoids formed by the P. aeruginosa phage Pf4 and Escherichia coli phage fd. A cryo-EM structure of the capsid from fd revealed distinct biochemical properties compared to Pf4. Fd and Pf4 formed tactoids with different morphologies that arise from differing phage geometries and packing densities, which in turn gave rise to different tactoid emergent properties. Finally, we showed that tactoids formed by either phage protect rod-shaped bacteria from antibiotic treatment, and that direct association with a tactoid is required for protection, demonstrating the formation of a diffusion barrier by the tactoid. This study provides insights into how filamentous molecules protect bacteria from extraneous substances in biofilms and in host-associated infections

Higher-order assemblies of oligomeric cargo receptor complexes form the membrane scaffold of the Cvt vesicle

Selective autophagy is the mechanism by which large cargos are specifically sequestered for degradation. The structural details of cargo and receptor assembly giving rise to autophagic vesicles remain to be elucidated. We utilize the yeast cytoplasm-to-vacuole targeting (Cvt) pathway, a prototype of selective autophagy, together with a multi-scale analysis approach to study the molecular structure of Cvt vesicles. We report the oligomeric nature of the major Cvt cargo Ape1 with a combined 2.8 Å X-ray and negative stain EM structure, as well as the secondary cargo Ams1 with a 6.3 Å cryo-EM structure. We show that the major dodecameric cargo prApe1 exhibits a tendency to form higher-order chain structures that are broken upon interaction with the receptor Atg19 in vitro The stoichiometry of these cargo-receptor complexes is key to maintaining the size of the Cvt aggregate in vivo Using correlative light and electron microscopy, we further visualize key stages of Cvt vesicle biogenesis. Our findings suggest that Atg19 interaction limits Ape1 aggregate size while serving as a vehicle for vacuolar delivery of tetrameric Ams1

The role of filamentous matrix molecules in shaping the architecture and emergent properties of bacterial biofilms.

Peer reviewed: TrueAcknowledgements: We would like to thank Kazune Tamura for advice on the initial plan of the manuscript, and Freya Bull as well as Zhexin (Eric) Wang for critical reading of the manuscript. T. A. M. B. would like to thank UKRI MRC (Programme MC_UP_1201/31), the EPSRC (Grant EP/V026623/1), the European Molecular Biology Organisation, the Leverhulme Trust and the Lister Institute for Preventative Medicine for support. For the purpose of open access, the MRC Laboratory of Molecular Biology has applied a CC BY public copyright licence to any Author Accepted Manuscript version arising.Publication status: PublishedNumerous bacteria naturally occur within spatially organised, multicellular communities called biofilms. Moreover, most bacterial infections proceed with biofilm formation, posing major challenges to human health. Within biofilms, bacterial cells are embedded in a primarily self-produced extracellular matrix, which is a defining feature of all biofilms. The biofilm matrix is a complex, viscous mixture primarily composed of polymeric substances such as polysaccharides, filamentous protein fibres, and extracellular DNA. The structured arrangement of the matrix bestows bacteria with beneficial emergent properties that are not displayed by planktonic cells, conferring protection against physical and chemical stresses, including antibiotic treatment. However, a lack of multi-scale information at the molecular level has prevented a better understanding of this matrix and its properties. Here, we review recent progress on the molecular characterisation of filamentous biofilm matrix components and their three-dimensional spatial organisation within biofilms

A role for Na+,K+-ATPase α1 in regulating Rab27a localisation on melanosomes

The mechanism(s) by which Rab GTPases are specifically recruited to distinct intracellular membranes remains elusive. Here we used Rab27a localisation onto melanosomes as a model to investigate Rab targeting. We identified the α1 subunit of Na+,K+-ATPase (ATP1a1) as a novel Rab27a interacting protein in melanocytes and showed that this interaction is direct with the intracellular M4M5 loop of ATP1a1 and independent of nucleotide bound status of the Rab. Knockdown studies in melanocytes revealed that ATP1a1 plays an essential role in Rab27a-dependent melanosome transport. Specifically, expression of ATP1a1, like the Rab27a GDP/GTP exchange factor (Rab3GEP), is essential for targeting and activation of Rab27a to melanosomes. Finally, we showed that the ability of Rab27a mutants to target to melanosomes correlates with the efficiency of their interaction with ATP1a1. Altogether these studies point to a new role for ATP1a1 as a regulator of Rab27a targeting and activation. © 2014 Booth et al

A role for Na+,K+-ATPase a1 in regulating Rab27a localisation on melanosomes (PLoS ONE (2014) 9:7 (e102851) DOI: 10.1371/journal. pone.0102851)

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Rab27a and MyoVa are the primary MIph interactors regulating melanosome transport in melanocytes

Melanosome transport in melanocytes is a model system for the study of cytoskeletal regulation of intracellular transport. Melanophilin (Mlph) is a Rab27a- and myosin Va (MyoVa)-binding protein that regulates this process. Using yeast two-hybrid screening, we identified MT plusend binding protein (EB1) as a melanocyte-expressed Mlph-interacting protein. To address the role of EB1 versus Rab27a and MyoVa interactions in Mlph targeting and function, we used siRNA and Mlph mutations to specifically disrupt each interaction in cultured melanocytes. Using the Mlph R35W mutant that blocks Mlph-Rab27a interaction and Rab27a siRNA we show this interaction is required for melanosome targeting and stability of Mlph. Mutants and siRNA that affect MlpMyoVa and Mlph-EB1 interactions reveal that while neither MyoVa nor EB1 affect Mlph targeting to melanosomes, MyoVa but not EB1 interaction is required for transport of melanosomes to peripheral dendrites. We propose that Mlph is targeted to and/or stabilised on melanosomes by Rab27a, and then recruits MyoVa, which provides additional stability to the complex and allows melanosomes to transfer from MT to actin-based transport and achieve peripheral distribution. EB1 appears to be non-essential to this process in cultured melanocytes, which suggests that it plays a redundant role and/or is required for melanocyte/keratinocyte contacts and melanosome transfer.publishersversionpublishe