Selective autophagy can be mediated via receptor molecules that link specific cargoes to the autophagosomal membranes decorated by ubiquitin-like microtubule-associated protein light chain 3 (LC3) modifiers. Although several autophagy receptors have been identified, little is known about mechanisms controlling their functions in vivo. In this work, we found that phosphorylation of an autophagy receptor, optineurin, promoted selective autophagy of ubiquitincoated cytosolic Salmonella enterica. The protein kinase TANK binding kinase 1 (TBK1) phosphorylated optineurin on serine-177, enhancing LC3 binding affinity and autophagic clearance of cytosolic Salmonella. Conversely, ubiquitin-or LC3-binding optineurin mutants and silencing of optineurin or TBK1 impaired Salmonella autophagy, resulting in increased intracellular bacterial proliferation. We propose that phosphorylation of autophagy receptors might be a general mechanism for regulation of cargo-selective autophagy.Macroautophagy (hereafter referred to as autophagy) is an evolutionarily conserved catabolic process by which cells deliver bulk cytosolic components for degradation to the lysosome (1-4). Selectivity in cargo targeting is mediated via autophagy receptors that simultaneously bind cargoes and autophagy modifiers, autophagy-related protein 8 (ATG8)/ microtubule-associated protein light chain 3 (LC3)/γ-aminobutyric acid receptor-associated protein (GABARAP) proteins, which are conjugated to the autophagosomal membranes (5, 6). The regulatory mechanisms controlling the spatiotemporal dynamics of the autophagy receptor-target interaction in cells remain unclear (7). Multiple autophagy receptors have been identified with the yeast two-hybrid system (8, 9), which included an N-terminal fragment of optineurin (OPTN), a ubiquitin-binding protein also known as NF-κB essential modulator-related protein ( Fig. 1, A and B). The specific interactions between OPTN and LC3/GABARAP proteins were verified by pull-down assays in mammalian cells, directed yeast two-hybrid transformations, and in vitro using purified proteins ( Fig. 1C and fig. S1, A and B) (10). OPTN bound to ubiquitin chains and autophagy modifiers ATG8/LC3/GABARAP proteins but not to mono-ubiquitin or other ubiquitin-like proteins ( Fig. 1C and fig. S1C). Deletion mapping of the N-terminal region of OPTN identified an LC3 interacting motif (LIR), a linear tetrapeptide sequence present in known autophagy receptors that binds directly to LC3/GABARAP modifiers (9, 11, 12). The LIR was located between the coiled-coil domains of OPTN encompassing amino acids 169 to 209 (Fig. 1A) and was essential for in vitro and in vivo binding between OPTN and LC3/ GABARAP (Fig. 1, B and C, and figs. S1A and S2A). Single point mutations at either OPTN Phe 178 →Ala 178 (F178A) or I181A (13), corresponding to the WxxL of p62, were sufficient to abrogate the interaction with LC3/GABARAP proteins, whereas these mutants were still able to bind to linear ubiquitin chains fused to glutathione S-transferase (GST-4xUb) (...

Amyotrophic lateral sclerosis (ALS) is a genetically heterogeneous neurodegenerative syndrome hallmarked by adult-onset loss of motor neurons. We performed exome sequencing of 252 familial ALS (fALS) and 827 control individuals. Gene-based rare variant analysis identified an exome-wide significant enrichment of eight loss-of-function (LoF) mutations in TBK1 (encoding TANK-binding kinase 1) in 13 fALS pedigrees. No enrichment of LoF mutations was observed in a targeted mutation screen of 1,010 sporadic ALS and 650 additional control individuals. Linkage analysis in four families gave an aggregate LOD score of 4.6. In vitro experiments confirmed the loss of expression of TBK1 LoF mutant alleles, or loss of interaction of the C-terminal TBK1 coiled-coil domain (CCD2) mutants with the TBK1 adaptor protein optineurin, which has been shown to be involved in ALS pathogenesis. We conclude that haploinsufficiency of TBK1 causes ALS and fronto-temporal dementia.

Selective autophagy of damaged mitochondria requires autophagy receptors optineurin (OPTN), NDP52 (CALCOCO2), TAX1BP1, and p62 (SQSTM1) linking ubiquitinated cargo to autophagic membranes. By using quantitative proteomics, we show that Tank-binding kinase 1 (TBK1) phosphorylates all four receptors on several autophagyrelevant sites, including the ubiquitin-and LC3-binding domains of OPTN and p62/SQSTM1 as well as the SKICH domains of NDP52 and TAX1BP1. Constitutive interaction of TBK1 with OPTN and the ability of OPTN to bind to ubiquitin chains are essential for TBK1 recruitment and kinase activation on mitochondria. TBK1 in turn phosphorylates OPTN's UBAN domain at S473, thereby expanding the binding capacity of OPTN to diverse Ub chains. In combination with phosphorylation of S177 and S513, this posttranslational modification promotes recruitment and retention of OPTN/TBK1 on ubiquitinated, damaged mitochondria. Moreover, phosphorylation of OPTN on S473 enables binding to pS65 Ub chains and is also implicated in PINK1-driven and Parkin-independent mitophagy. Thus, TBK1-mediated phosphorylation of autophagy receptors creates a signal amplification loop operating in selective autophagy of damaged mitochondria.A s a cell survival pathway, autophagy selectively frees the cytosolic compartment from bulky protein aggregates, invading bacteria or damaged organelles such as mitochondria and peroxisomes (1, 2). In this context, the posttranslational modifier ubiquitin (Ub) has been widely recognized as a selective signal driving autophagy of such cellular components and cargoes (3, 4). Recently, ubiquitin itself has been discovered to be phosphorylated to promote autophagic clearance of damaged mitochondria (mitophagy; reviewed in refs. 5 and 6). Ser/Thr kinase PINK1 phosphorylates S65 of Ub, which is critical for two steps of this process: allosteric activation of the E3 Ub ligase Parkin and recruitment of the autophagic machinery, including autophagy receptors (7)(8)(9)(10)(11)(12)(13)(14).Autophagy receptors function as decoders for the various ubiquitin signals on cargoes, linking cargoes to autophagosomal membranes (4); however, the basis of their individual recruitment to cargo as well as their distinct and cooperative functions in cargo sequestration are still poorly understood. The autophagy receptors optineurin (OPTN) and p62 are first activated by protein kinases to effectively target autophagic membranes or their polyUb cargo (15-17). TANK-binding kinase 1 (TBK1) phosphorylates OPTN on S177, thereby enhancing LC3-binding affinity and autophagic clearance of cytosolic Salmonella (15). Activity and specificity of TBK1 are defined by adaptor proteins; these recruit TBK1 to microdomains on ubiquitinated Salmonella or mitochondria, thereby facilitating its local clustering and activation (18), where it in turn can phosphorylate autophagy receptors (15). It is relevant to stress that a number of mutations in both OPTN and TBK1 have been identified in patients suffering from amyotrophic lateral sclerosis (ALS) and...

Cell behavior is governed by interactions with the cellular environment. [1][2][3] These interactions include cell-cell as well as cell-extracellular matrix (ECM) contacts and act, in addition to soluble growth factors, as key regulators of cell survival, proliferation, and differentiation. However, not only the molecular composition of the contact sites, but also their spatial distributions impact cell behavior. [ 4 , 5 ] Realizing cell-culture scaffolds that mirror the complex in vivo arrangement of ECM components is an active area of biomaterials engineering. Corresponding 2D lithographically defi ned micropatterned structures are widely used and have even become commercially available. [6][7][8][9] In addition to patterned ligand distributions, mechanical interactions between cells and their environment also play an important role in regulating cellular functions. [ 10 ] Consequently, corresponding fl exible substrates were developed that allow measurements of cellular forces in 2D on planar substrates or pillar arrays. [ 11 , 12 ] However, discrepancies between cell behavior in vivo and in artifi cial 2D environments have become evident. [ 5 , 13 , 14 ] Therefore, devices that capture more of the structural complexity present in 3D tissues are highly desirable.Fibrous collagen or matrigel matrices are commonly used to study 3D cell behavior, [ 15 , 16 ] but these matrices have a random pore size and are structurally and chemically ill-defi ned. We [ 17 ] and others [18][19][20] have recently shown that direct laser writing (DLW) is a versatile technique to fabricate tailored 3D cell-culture scaffolds in the micrometer to nanometer range. By using an adequate photoresist, elastic 3D scaffolds for cell force measurements have also been realized. [ 17 ] These DLW scaffolds have been homogeneously coated with ECM molecules. Ideally, they should have an adjustable distribution of cell-substrate contact sites to manipulate cell adhesion and cell shape in all three dimensions. In this communication, we report on tailored 3D scaffolds with a controlled ECM distribution. By sequential DLW of two different photoresists, composite-polymer scaffolds with distinct protein-binding properties are fabricated and selectively biofunctionalized thereafter. We further demonstrate that cells cultured in these scaffolds selectively form cell adhesion sites with the functionalized parts, allowing for control of cell adhesion and cell shape in three dimensions, forming the basis for future designer tissue-culture scaffolds.To fabricate micrometer-scale composite-polymer scaffolds with distinct protein-binding properties as depicted in Figure 1 , we fi rst selected and characterized appropriate materials. For the small protein-binding cubes in the scaffolds we selected Ormocomp, a member of the inorganic (Si-O-Si)-organic hybrid polymer Ormocer family (Fraunhofer Institute for Silica Research, Würzburg, Germany). Ormocomp is a biocompatible photoresist that has previously been used in our lab to manufacture elastic 3D scaffolds...

3D printing is a powerful emerging technology for the tailored fabrication of advanced functional materials. This Review summarizes the state-of-the art with regard to 3D laser micro- and nanoprinting and explores the chemical challenges limiting its full exploitation: from the development of advanced functional materials for applications in cell biology and electronics to the chemical barriers that need to be overcome to enable fast writing velocities with resolution below the diffraction limit. We further explore chemical means to enable direct laser writing of multiple materials in one resist by highly wavelength selective (λ-orthogonal) photochemical processes. Finally, chemical processes to construct adaptive 3D written structures that are able to respond to external stimuli, such as light, heat, pH value, or specific molecules, are highlighted, and advanced concepts for degradable scaffolds are explored.

Cells in physiological 3D environments differ considerably in morphology and differentiation from those in 2D tissue culture. Naturally derived polymer systems are frequently used to study cells in 3D. These 3D matrices are complex with respect to their chemical composition, mechanical properties, and geometry. Therefore, there is a demand for well-defined 3D scaffolds to systematically investigate cell behavior in 3D. Here, fabrication techniques, materials, architectures, biochemical functionalizations, and mechanical properties of 3D scaffolds are discussed. In particular, work focusing on single cells and small cell assemblies grown in tailored synthetic 3D scaffolds fabricated by computer-based techniques are reviewed and the influence of these environments on cell behavior is evaluated.

The combination of three different photoresists into a single direct laser written 3D microscaffold permits functionalization with two bioactive full-length proteins. The cell-instructive microscaffolds consist of a passivating framework equipped with light activatable constituents featuring distinct protein-binding properties. This allows directed cell attachment of epithelial or fibroblast cells in 3D.

The host endolysosomal compartment is often manipulated by intracellular bacterial pathogens. Salmonella (Salmonella enterica serovar Typhimurium) secrete numerous effector proteins, including SifA, through a specialized type III secretion system to hijack the host endosomal system and generate the Salmonella-containing vacuole (SCV). To form this replicative niche, Salmonella targets the Rab7 GTPase to recruit host membranes through largely unknown mechanisms. We show that Pleckstrin homology domain-containing protein family member 1 (PLEKHM1), a lysosomal adaptor, is targeted by Salmonella through direct interaction with SifA. By binding the PLEKHM1 PH2 domain, Salmonella utilize a complex containing PLEKHM1, Rab7, and the HOPS tethering complex to mobilize phagolysosomal membranes to the SCV. Depletion of PLEKHM1 causes a profound defect in SCV morphology with multiple bacteria accumulating in enlarged structures and significantly dampens Salmonella proliferation in multiple cell types and mice. Thus, PLEKHM1 provides a critical interface between pathogenic infection and the host endolysosomal system.