Autocatalytic, bistable, oscillatory networks of biologically relevant organic reactions.

Networks of organic chemical reactions are important in life and probably played a central part in its origin. Network dynamics regulate cell division, circadian rhythms, nerve impulses and chemotaxis, and guide the development of organisms. Although out-of-equilibrium networks of chemical reactions have the potential to display emergent network dynamics such as spontaneous pattern formation, bistability and periodic oscillations, the principles that enable networks of organic reactions to develop complex behaviours are incompletely understood.

Size, organization, and dynamics of soluble SQSTM1 and LC3-SQSTM1 complexes in living cells.

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.

Nuclear LC3 Associates with Slowly Diffusing Complexes that Survey the Nucleolus.

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.

Microtubule motors power plasma membrane tubulation in clathrin-independent endocytosis.

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.

Size, stoichiometry, and organization of soluble LC3-associated complexes.

MAP1LC3B, an ortholog of yeast Atg8 and a member of the family of proteins formerly also known as ATG8 in mammals (LC3B henceforth in the text), 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.

Motor and tail homology 1 (Th1) domains antagonistically control myosin-1 dynamics.

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.

Overexpression of caveolin-1 is sufficient to phenocopy the behavior of a disease-associated mutant.

Mutations and alterations in caveolin-1 expression levels have been linked to a number of human diseases. How misregulation of caveolin-1 contributes to disease is not fully understood, but has been proposed to involve the intracellular accumulation of mutant forms of the protein. To better understand the molecular basis for trafficking defects that trap caveolin-1 intracellularly, we compared the properties of a GFP-tagged version of caveolin-1 P132L, a mutant form of caveolin-1 previously linked to breast cancer, with wild-type caveolin-1.

Analysis of protein and lipid dynamics using confocal fluorescence recovery after photobleaching (FRAP).

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.

Imaging protein complex formation in the autophagy pathway: analysis of the interaction of LC3 and Atg4B(C74A) in live cells using Förster resonance energy transfer and fluorescence recovery after photobleaching.

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.