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Selective macroautophagy/autophagy—with the help of molecular receptors—captures cargo for lysosomal degradation. Among the best-studied molecular receptors is SQSTM1/p62, a homo-oligomeric ubiquitin binding protein, which binds to both cargo and MAP1LC3B/LC3, a protein important for autophagosome biogenesis. Although the mechanisms underlying interaction of LC3 and SQSTM1 have been extensively studied, very little is known about the size or organization of soluble complexes formed between SQSTM1 and LC3 prior to phagophore (the autophagosome precursor) binding in live cells at the molecular level. To address this question, in the current study we use a combination of 2 microscopy-based approaches, FRET microscopy and confocal FRAP, to study the nanoscale properties of soluble SQSTM1 complexes and SQSTM1-LC3 complexes in living HeLa cells. We find that, independent of puncta, SQSTM1 oligomerizes to form very slowly diffusing complexes that contain multiple copies of SQSTM1 within FRET proximity of one another. Furthermore, we show that the interactions of soluble pools of LC3 and SQSTM1 can be readily detected by both FRAP and FRET. Finally, we uncover unexpected roles of SQSTM1's PB1 domain, a region of the protein involved in homo-oligomer formation, in complex formation. Taken together, these findings provide new insights into the nature of nanometer-sized protein complexes in the autophagy pathway.

A few-component network of biologically relevant, organic reactions displays bistability and oscillations, without an enzymatic catalyst. Dissipative chemical reaction networks are systems that operate away from equilibrium and exhibit features such as continuous regeneration of components and autonomous regulation. The biological cell is one such out-of-equilibrium chemical network. Until now it has not been possible to recreate this type of dynamic behavior using simple organic molecules relevant to prebiotic Earth. Now, George Whitesides and colleagues demonstrate a few-component system of interacting organic species that, combined into a reaction network, displays autocatalytic and oscillatory features. All of the organic components are relatively simple and do not require enzymatic catalysis to react. Networks of organic chemical reactions are important in life and probably played a central part in its origin1,2,3. Network dynamics regulate cell division4,5,6, circadian rhythms7, nerve impulses8 and chemotaxis9, and guide the development of organisms10. Although out-of-equilibrium networks of chemical reactions have the potential to display emergent network dynamics11 such as spontaneous pattern formation, bistability and periodic oscillations12,13,14, the principles that enable networks of organic reactions to develop complex behaviours are incompletely understood. Here we describe a network of biologically relevant organic reactions (amide formation, thiolate–thioester exchange, thiolate–disulfide interchange and conjugate addition) that displays bistability and oscillations in the concentrations of organic thiols and amides. Oscillations arise from the interaction between three subcomponents of the network: an autocatalytic cycle that generates thiols and amides from thioesters and dialkyl disulfides; a trigger that controls autocatalytic growth; and inhibitory processes that remove activating thiol species that are produced during the autocatalytic cycle. In contrast to previous studies that have demonstrated oscillations and bistability using highly evolved biomolecules (enzymes15 and DNA16,17) or inorganic molecules of questionable biochemical relevance (for example, those used in Belousov–Zhabotinskii-type reactions)18,19, the organic molecules we use are relevant to metabolism and similar to those that might have existed on the early Earth. By using small organic molecules to build a network of organic reactions with autocatalytic, bistable and oscillatory behaviour, we identify principles that explain the ways in which dynamic networks relevant to life could have developed. Modifications of this network will clarify the influence of molecular structure on the dynamics of reaction networks, and may enable the design of biomimetic networks and of synthetic self-regulating and evolving chemical systems.

Multianalyte microphysiometry, a real-time instrument for simultaneous measurement of metabolic analytes in a microfluidic environment, was used to explore the effects of cholera toxin (CTx). Upon exposure of CTx to PC-12 cells, anaerobic respiration was triggered, measured as increases in acid and lactate production and a decrease in the oxygen uptake. We believe the responses observed are due to a CTx-induced activation of adenylate cyclase, increasing cAMP production and resulting in a switch to anaerobic respiration. Inhibitors (H-89, brefeldin A) and stimulators (forskolin) of cAMP were employed to modulate the CTx-induced cAMP responses. The results of this study show the utility of multianalyte microphysiometry to quantitatively determine the dynamic metabolic effects of toxins and affected pathways.

MAP1LC3B (microtubule-associated protein 1 light chain 3, LC3) is a key component of the autophagy pathway, contributing to both cargo selection and autophagosome formation in the cytoplasm. Emerging evidence suggests that nuclear forms of LC3 are also functionally important; however, the mechanisms that facilitate the nuclear targeting and trafficking of LC3 between the nucleus and cytoplasm under steady-state conditions are poorly understood. In this study, we examine how residues known to regulate the interactions between LC3 and other proteins or RNA (F52 L53, R68-R70 and G120) contribute to its nuclear targeting, nucleocytoplasmic transport and association with nucleoli and other nuclear components. We find that residues F52 L53 and R68-70, but not G120, regulate targeting of LC3 to the nucleus, its rates of nucleocytoplasmic transport and the apparent sizes of LC3-associated complexes in the nucleus inferred from fluorescence recovery after photobleaching (FRAP) measurements. We also show that LC3 is enriched in nucleoli and its triple arginine motif is especially important for nucleolar targeting. Finally, we identify a series of candidate nuclear LC3-interacting proteins using mass spectrometry, including MAP1B, tubulin and several 40S ribosomal proteins. These findings suggest LC3 is retained in the nucleus in association with high-molecular weight complexes that continuously scan the nucleolus.

How the plasma membrane is bent to accommodate clathrin-independent endocytosis remains uncertain. Recent studies suggest Shiga and cholera toxin induce membrane curvature required for their uptake into clathrin-independent carriers by binding and cross-linking multiple copies of their glycosphingolipid receptors on the plasma membrane. But it remains unclear if toxin-induced sphingolipid crosslinking provides sufficient mechanical force for deforming the plasma membrane, or if host cell factors also contribute to this process. To test this, we imaged the uptake of cholera toxin B-subunit into surface-derived tubular invaginations. We found that cholera toxin mutants that bind to only one glycosphingolipid receptor accumulated in tubules, and that toxin binding was entirely dispensable for membrane tubulations to form. Unexpectedly, the driving force for tubule extension was supplied by the combination of microtubules, dynein and dynactin, thus defining a novel mechanism for generating membrane curvature during clathrin-independent endocytosis.

Class 1 myosins are monomeric motor proteins that fulfill diverse functions at the membrane/cytoskeletal interface. All myosins-1 contain a motor domain, which binds actin, hydrolyzes ATP, and generates forces, and a TH1 domain, which interacts directly with membrane lipids. In most cases, TH1 is needed for proper subcellular localization and presumably function, although little is known about how this domain regulates the behavior of class 1 myosins in live cells. To address this, we used single molecule total internal reflection fluorescence microscopy to examine the dynamics of the well-characterized myosin-1a isoform during interactions with the cortex of living cells. Our studies revealed that full-length myosin-1a exhibits restricted mobility relative to TH1 alone. Motor domain mutations that disrupt actin binding increased the mobility of full-length myosin-1a, whereas mutations to the TH1 domain that are known to reduce steady-state targeting to the plasma membrane unexpectedly reduced mobility. Deletion of the calmodulin-binding lever arm in Myo1a mimicked the impact of actin-binding mutations. Finally, myosin-1b, which demonstrates exquisite sensitivity to mechanical load, exhibited dynamic behavior nearly identical to myosin-1a. These studies are the first, to our knowledge, to explore class 1 myosin dynamics at the single-molecule level in living cells; our results suggest a model where the motor domain restricts dynamics via a mechanism that requires the lever arm, whereas the TH1 domain allows persistent diffusion in close proximity to the plasma membrane.

LC3 is a key component of the autophagy pathway, where it functions in autophagosome formation and cargo recruitment. Recent proteomics analysis indicates LC3 and other members of the ATG8 family interact with 67 different proteins, many of which appear to bind to common sites on LC3. However, it is currently unknown how many proteins are bound to LC3 at a given time, or if cytoplasmic and nuclear forms of LC3 interact with similar or different proteins. Here, we report several independent lines of evidence that suggest LC3 is constitutively associated with a high molecular weight complex in both the cytoplasm and nucleus. First, confocal FRAP measurements indicate that Venus-LC3 diffuses significantly more slowly than Venus alone in both the cytoplasm and nucleus of living cells. Second, fluorescence correlation spectroscopy measurements of freshly prepared cell homogenates report the correlation time of Venus-LC3 is longer than that of Venus alone. Third, both cytoplasmic and nuclear forms of endogenous LC3 and Venus-LC3 migrate as part of a high molecular weight complex in blue native gel electrophoresis. To test whether multiple copies of LC3 are contained within the same complex, we performed FRET analysis between Venus- and Cerulean-tagged LC3, homoFRET analysis of Venus-LC3, and brightness analysis of Venus-LC3. No FRET was detected between LC3 molecules in the cytoplasm or in cell homogenates, and the brightness of LC3 was consistent with approximately 1 Venus-LC3 per complex. However, a small level of FRET was measured between LC3 molecules in the nucleus, suggesting multiple copies of the protein may be present within the same complex. Experiments are currently underway to determine how the formation of these complexes is regulated as well as to identify other complex components.

Fluorescence recovery after photobleaching (FRAP) is a powerful, versatile, and widely accessible tool to monitor molecular dynamics in living cells that can be performed using modern confocal microscopes. Although the basic principles of FRAP are simple, quantitative FRAP analysis requires careful experimental design, data collection, and analysis. In this unit, we discuss the theoretical basis for confocal FRAP, followed by step-by-step protocols for FRAP data acquisition using a laser-scanning confocal microscope for (1) measuring the diffusion of a membrane protein, (2) measuring the diffusion of a soluble protein, and (3) analysis of intracellular trafficking. Finally, data analysis procedures are discussed, and an equation for determining the diffusion coefficient of a molecular species undergoing pure diffusion is presented.

Microtubule associated protein 1 light chain 3B (LC3/ATG8) functions in autophagosome formation and autophagy substrate recruitment. LC3 exists in both a soluble (autophagosome-independent) form as well as a lipid modified form that becomes tightly incorporated into autophagosomal membranes. Although LC3 is known to associate with tens of proteins, relatively little is known about soluble LC3 aside from its interactions with the LC3 lipid conjugation machinery. In previous studies we found autophagosome-independent GFP-LC3 diffuses unusually slowly for a protein of its size, suggesting it may be constitutively associated with a high molecular weight complex, form homo-oligomers or aggregates, or reversibly bind microtubules or membranes. To distinguish between these possibilities, we characterized the size, stoichiometry, and organization of autophagosome-independent LC3 in living cells and in cytoplasmic extracts using Fluorescence Recovery After Photobleaching (FRAP) and Fluorescence Polarization Fluctuation Analysis (FPFA). We found that the diffusion of autophagosome-independent LC3 was unaffected by either mutational disruption of its lipid modification or microtubule depolymerization, suggesting this form of LC3 does not reversibly bind to microtubules or membranes. Brightness and homoFRET analysis indicate LC3 does not homo-oligomerize, ruling out this as a possibility for its slow diffusion. In contrast, mutation of specific residues on LC3 required for binding other proteins and mRNA led to changes in the effective hydrodynamic radius of the protein as well as its stoichiometry. This suggests that LC3 associates with a multi-component complex consisting of either proteins or RNA. We conclude that autophagosome-independent LC3 associates with a complex with an effective size of ∼500 kDa in the cytoplasm. These findings provide new insights into the nature of autophagosome-independent LC3 and illustrate the power of FRAP and FPFA to provide novel insights into the emergent properties of protein complexes in the autophagy pathway.

Extended abstract of a paper presented at Microscopy and Microanalysis 2011 in Nashville, Tennessee, USA, August 7–August 11, 2011.

The protein microtubule-associated protein 1, light chain 3 (LC3) functions in autophagosome formation and plays a central role in the autophagy pathway. Previously, we found LC3 diffuses more slowly in cells than is expected for a freely diffusing monomer, suggesting it may constitutively associate with a macromolecular complex containing other protein components of the pathway. In the current study, we used Förster resonance energy transfer (FRET) microscopy and fluorescence recovery after photobleaching (FRAP) to investigate the interactions of LC3 with Atg4B(C74A), a catalytically inactive mutant of the cysteine protease involved in lipidation and de-lipidation of LC3, as a model system to probe protein complex formation in the autophagy pathway. We show Atg4B(C74A) is in FRET proximity with LC3 in both the cytoplasm and nucleus of living cells, consistent with previous biochemical evidence that suggests these proteins directly interact. In addition, overexpressed Atg4B(C74A) diffuses significantly more slowly than predicted based on its molecular weight, and its translational diffusion coefficient is significantly slowed upon coexpression with LC3 to match that of LC3 itself. Taken together, these results suggest Atg4B(C74A) and LC3 are contained within the same multiprotein complex and that this complex exists in both the cytoplasm and nucleoplasm of living cells.

Autophagy is a lysosomal degradation pathway that is important for the maintenance of cellular homeostasis. Although the formation of autophagosomes occurs in the cytoplasm, it is increasingly evident that several key proteins in the autophagy pathway shuttle in and out of the nucleus. We previously reported that LC3, a protein that participates in autophagosome formation and cargo selection, is enriched in the nucleus in a slowly diffusing form. However, the mechanisms that retain LC3 in the nucleus and control its nuclear dynamics remain poorly understood. To address this, we used a combination of fluorescence microscopy and FRAP to study Venus-LC3 in HeLa cells. We find that mutating residues involved in binding of LC3 to other proteins and RNA moderately decreases its nucleocytoplasmic ratio, but disrupting LC3 lipid modification does not change its nucleocytoplasmic ratio. This suggests that the majority of LC3 in the nucleus is soluble, and it is retained in the nucleus by interactions with either proteins or RNA. Consistent with this, soluble nuclear LC3 diffuses more slowly than predicted for a monomer. Perturbing the autophagy pathway with rapamycin or chloroquine has little effect on LC3's nuclear localization, but does change the apparent size of LC3-associated complexes. This implies that soluble nuclear LC3 associates with complexes with functions possibly related to autophagy. Unexpectedly, we also find that nuclear LC3 is enriched within the nucleolus. We show that the triple arginine motif of LC3, a region previously shown to bind to both proteins and RNA, shares similarities with a consensus nucleolar detention sequence. Furthermore, perturbation of LC3's triple arginine motif completely abolishes its localization to the nucleolus. Together, these findings reveal a potential role for both protein-protein and protein-RNA interactions in targeting LC3 to the nucleus and nucleolus.