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

Numerous 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.

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.

Architecture of the biofilm-associated archaic Chaperone-Usher pilus CupE from Pseudomonas aeruginosa.

Chaperone-Usher Pathway (CUP) pili are major adhesins in Gram-negative bacteria, mediating bacterial adherence to biotic and abiotic surfaces. While classical CUP pili have been extensively characterized, little is known about so-called archaic CUP pili, which are phylogenetically widespread and promote biofilm formation by several human pathogens. In this study, we present the electron cryomicroscopy structure of the archaic CupE pilus from the opportunistic human pathogen Pseudomonas aeruginosa. We show that CupE1 subunits within the pilus are arranged in a zigzag architecture, containing an N-terminal donor ß-strand extending from each subunit into the next, where it is anchored by hydrophobic interactions, with comparatively weaker interactions at the rest of the inter-subunit interface. Imaging CupE pili on the surface of P. aeruginosa cells using electron cryotomography shows that CupE pili adopt variable curvatures in response to their environment, which might facilitate their role in promoting cellular attachment. Finally, bioinformatic analysis shows the widespread abundance of cupE genes in isolates of P. aeruginosa and the co-occurrence of cupE with other cup clusters, suggesting interdependence of cup pili in regulating bacterial adherence within biofilms. Taken together, our study provides insights into the architecture of archaic CUP pili, providing a structural basis for understanding their role in promoting cellular adhesion and biofilm formation in P. aeruginosa.

Identifying proteins bound to native mitotic ESC chromosomes reveals chromatin repressors are important for compaction.

Epigenetic information is transmitted from mother to daughter cells through mitosis. Here, to identify factors that might play a role in conveying epigenetic memory through cell division, we report on the isolation of unfixed, native chromosomes from metaphase-arrested cells using flow cytometry and perform LC-MS/MS to identify chromosome-bound proteins. A quantitative proteomic comparison between metaphase-arrested cell lysates and chromosome-sorted samples reveals a cohort of proteins that were significantly enriched on mitotic ESC chromosomes. These include pluripotency-associated transcription factors, repressive chromatin-modifiers such as PRC2 and DNA methyl-transferases, and proteins governing chromosome architecture. Deletion of PRC2, Dnmt1/3a/3b or Mecp2 in ESCs leads to an increase in the size of individual mitotic chromosomes, consistent with de-condensation. Similar results were obtained by the experimental cleavage of cohesin. Thus, we identify chromosome-bound factors in pluripotent stem cells during mitosis and reveal that PRC2, DNA methylation and Mecp2 are required to maintain chromosome compaction.

Phage liquid crystalline droplets form occlusive sheaths that encapsulate and protect infectious rod-shaped bacteria.

The opportunistic pathogen Pseudomonas aeruginosa is a major cause of antibiotic-tolerant infections in humans. P. aeruginosa evades antibiotics in bacterial biofilms by up-regulating expression of a symbiotic filamentous inoviral prophage, Pf4. We investigated the mechanism of phage-mediated antibiotic tolerance using biochemical reconstitution combined with structural biology and high-resolution cellular imaging. We resolved electron cryomicroscopy atomic structures of Pf4 with and without its linear single-stranded DNA genome, and studied Pf4 assembly into liquid crystalline droplets using optical microscopy and electron cryotomography. By biochemically replicating conditions necessary for antibiotic protection, we found that phage liquid crystalline droplets form phase-separated occlusive compartments around rod-shaped bacteria leading to increased bacterial survival. Encapsulation by these compartments was observed even when inanimate colloidal rods were used to mimic rod-shaped bacteria, suggesting that shape and size complementarity profoundly influences the process. Filamentous inoviruses are pervasive across prokaryotes, and in particular, several Gram-negative bacterial pathogens including Neisseria meningitidis, Vibrio cholerae, and Salmonella enterica harbor these prophages. We propose that biophysical occlusion mediated by secreted filamentous molecules such as Pf4 may be a general strategy of bacterial survival in harsh environments.

The Small Non-coding Vault RNA1-1 Acts as a Riboregulator of Autophagy.

Vault RNAs (vtRNA) are small non-coding RNAs transcribed by RNA polymerase III found in many eukaryotes. Although they have been linked to drug resistance, apoptosis, and viral replication, their molecular functions remain unclear. Here, we show that vault RNAs directly bind the autophagy receptor sequestosome-1/p62 in human and murine cells. Overexpression of human vtRNA1-1 inhibits, while its antisense LNA-mediated knockdown enhances p62-dependent autophagy. Starvation of cells reduces the steady-state and p62-bound levels of vault RNA1-1 and induces autophagy. Mechanistically, p62 mutants that fail to bind vtRNAs display increased p62 homo-oligomerization and augmented interaction with autophagic effectors. Thus, vtRNA1-1 directly regulates selective autophagy by binding p62 and interference with oligomerization, a critical step of p62 function. Our data uncover a striking example of the potential of RNA to control protein functions directly, as previously recognized for protein-protein interactions and post-translational modifications.

Recombinant Expression, Purification, and Assembly of p62 Filaments.

This chapter describes the recombinant overexpression of the canonical selective autophagy receptor p62/SQSTM1 in E. coli and affinity purification. Also described is the method to induce p62 filament assembly and their visualization by negative stain electron microscopy (EM). In cells, p62 forms large structures termed p62 bodies and has been shown to be aggregation prone. This tendency to aggregate poses problems for expression and purification in vitro, which is a prerequisite for structural analysis. Here, we describe the method to express and purify soluble p62, using the solubility tag, MBP, in conjunction with autoinduction. Furthermore, we describe the protocol to assemble p62 into filaments by controlling the ionic strength of its buffer, as well as the preparation of negative stain EM grids to visualize the filaments. In vitro formed p62 filaments can be used to study receptor cargo interactions in minimal reconstituted autophagy model systems.

Phasing out the bad-How SQSTM1/p62 sequesters ubiquitinated proteins for degradation by autophagy.

The degradation of misfolded, ubiquitinated proteins is essential for cellular homeostasis. These proteins are primarily degraded by the ubiquitin-proteasome system (UPS) and macroautophagy/autophagy serves as a backup mechanism when the UPS is overloaded. How autophagy and the UPS are coordinated is not fully understood. During the autophagy of misfolded, ubiquitinated proteins, referred to as aggrephagy, substrate proteins are clustered into larger structures in a SQSTM1/p62-dependent manner before they are sequestered by phagophores, the precursors to autophagosomes. We have recently shown that SQSTM1/p62 and ubiquitinated proteins spontaneously phase separate into micrometer-sized clusters in vitro. This enabled us to characterize the properties of the ubiquitin-positive substrates that are necessary for the SQSTM1/p62-mediated cluster formation. Our results suggest that aggrephagy is triggered by the accumulation of substrates with multiple ubiquitin chains and that the process can be inhibited by active proteasomes.

p62 filaments capture and present ubiquitinated cargos for autophagy.

The removal of misfolded, ubiquitinated proteins is an essential part of the protein quality control. The ubiquitin-proteasome system (UPS) and autophagy are two interconnected pathways that mediate the degradation of such proteins. During autophagy, ubiquitinated proteins are clustered in a p62-dependent manner and are subsequently engulfed by autophagosomes. However, the nature of the protein substrates targeted for autophagy is unclear. Here, we developed a reconstituted system using purified components and show that p62 and ubiquitinated proteins spontaneously coalesce into larger clusters. Efficient cluster formation requires substrates modified with at least two ubiquitin chains longer than three moieties and is based on p62 filaments cross-linked by the substrates. The reaction is inhibited by free ubiquitin, K48-, and K63-linked ubiquitin chains, as well as by the autophagosomal marker LC3B, suggesting a tight cross talk with general proteostasis and autophagosome formation. Our study provides mechanistic insights on how substrates are channeled into autophagy.

Melanin Transferred to Keratinocytes Resides in Nondegradative Endocytic Compartments.

Melanin transfer from melanocytes to keratinocytes and subsequent accumulation in the supranuclear region is a critical process in skin pigmentation and protection against UVR. 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-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. After 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.

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 selective autophagy receptor p62 forms a flexible filamentous helical scaffold.

The 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.

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.

Rab11b mediates melanin transfer between donor melanocytes and acceptor keratinocytes via coupled exo/endocytosis.

The transfer of melanin from melanocytes to keratinocytes is a crucial process underlying maintenance of skin pigmentation and photoprotection against UV damage. Here, we present evidence supporting coupled exocytosis of the melanin core, or melanocore, by melanocytes and subsequent endocytosis by keratinocytes as a predominant mechanism of melanin transfer. Electron microscopy analysis of human skin samples revealed three lines of evidence supporting this: (1) the presence of melanocores in the extracellular space; (2) within keratinocytes, melanin was surrounded by a single membrane; and (3) this membrane lacked the melanosomal membrane protein tyrosinase-related protein 1 (TYRP1). Moreover, co-culture of melanocytes and keratinocytes suggests that melanin exocytosis is specifically induced by keratinocytes. Furthermore, depletion of Rab11b, but not Rab27a, caused a marked decrease in both keratinocyte-stimulated melanin exocytosis and transfer to keratinocytes. Thus, we propose that the predominant mechanism of melanin transfer is keratinocyte-induced exocytosis, mediated by Rab11b through remodeling of the melanosome membrane, followed by subsequent endocytosis by keratinocytes.

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.

Myrip uses distinct domains in the cellular activation of myosin VA and myosin VIIA in melanosome transport.

Myrip is a Rab27a and MyosinVIIa (MyoVIIa) linking protein that may regulate melanosome transport in the retinal pigment epithelium (RPE). Myrip also binds MyosinVa (MyoVa) in vitro however it is unclear whether this interaction is of sufficient affinity to be physiologically relevant. Here, we addressed the questions of whether Myrip interacts with MyoVa in cells and the molecular basis of cellular activation of MyoVa and MyoVIIa by Myrip. To answer these questions we used melanosome transport in skin melanocytes and RPE cells as read-outs of MyoVa and MyoVIIa activity. We found that Myrip recruits and activates MyoVa on skin melanosomes with similar efficiency to the established MyoVa activator Melanophilin (Mlph). Mutagenesis showed that a Myrip-Mlph conserved amphipathic helix (MMAH) is essential for MyoVa interaction while other Myrip regions, including the MyoVa exon F binding domain equivalent, play non-essential roles in this interaction. This suggests that, in contrast to Mlph, Myrip interacts with MyoVa lacking melanocyte-specific exon F. Parallel studies of RPE melanosome transport reveal that Myrip-specific inserts, but not the MMAH, are essential for MyoVIIa activation. We conclude that Myrip is a versatile Rab27a-associated myosin-activating protein that mediates cellular activation of MyoVa and MyoVIIa via non-overlapping domains.

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.

Rab27a and MyoVa are the primary Mlph 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 plus-end 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 Mlph-MyoVa 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.