Lewis J. Kraft

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. Finally, data analysis procedures are discussed, and an equation for determining the diffusion coefficient of a molecular species undergoing pure diffusion is presented. Curr. Protoc. Cytom. 62:2.19.1‐2.19.29.

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. 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.

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. Unexpectedly, wild‐type caveolin‐1‐GFP also accumulated intracellularly, leading us to examine the mechanisms underlying the abnormal localization of the wild type and mutant protein in more detail. We show that both the nature of the tag and cellular context impact the subcellular distribution of caveolin‐1, demonstrate that even the wild‐type form of caveolin‐1 can function as a dominant negative under some conditions, and identify specific conformation changes associated with incorrectly targeted forms of the protein. In addition, we find intracellular caveolin‐1 is phosphorylated on Tyr14, but phosphorylation is not required for mistrafficking of the protein. These findings identify novel properties of mistargeted forms of caveolin‐1 and raise the possibility that common trafficking defects underlie diseases associated with overexpression and mutations in caveolin‐1.

Imaging protein complex formation in the autophagy pathway: analysis of the interaction of LC3 and Atg4BC74A 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. 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.

The Effects of Cholera Toxin on Cellular Energy Metabolism

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.

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. In previous studies we found autophagosome-independent GFP-LC3B diffuses unusually slowly for a protein of its size, suggesting it may constitutively associate 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 LC3B 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 LC3B was unaffected by either mutational disruption of its lipid modification or microtubule depolymerization. Brightness and homo-FRET analysis indicate LC3B does not homo-oligomerize. However, mutation of specific residues on LC3B required for binding other proteins and mRNA altered the effective hydrodynamic radius of the protein as well as its stoichiometry. We conclude that when not bound to autophagosomes, LC3B associates with a multicomponent complex with an effective size of ~500 kDa in the cytoplasm. These findings provide new insights into the nature of soluble LC3B and illustrate the power of FRAP and FPFA to investigate the emergent properties of protein complexes in the autophagy pathway.

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. 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.

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. 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.

Microtubule Motors Drive Plasma Membrane Tubulation in Clathrin-Independent Endocytosis

How the plasma membrane is bent to accommodate clathrin-independent endocytosis is poorly understood. Recent studies suggest the exogenous clathrin independent cargo molecules Shiga toxin and cholera toxin induce the negative membrane curvature required for endocytic uptake 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-attached tubular invaginations in live cells. We found that a cholera toxin mutant that binds to only one glycosphingolipid receptor accumulates in tubules, and that toxin binding is entirely dispensable for membrane tubulations to form. Unexpectedly, the driving force for tubule extension was found to be supplied by the combination of microtubules, dynein, and dynactin, thus defining a novel mechanism for generation or extension of membrane curvature during endocytic uptake at the plasma membrane.