The MYO6 interactome reveals adaptor complexes coordinating early endosome and cytoskeletal dynamics.

The intracellular functions of myosin motors requires a number of adaptor molecules, which control cargo attachment, but also fine-tune motor activity in time and space. These motor-adaptor-cargo interactions are often weak, transient or highly regulated. To overcome these problems, we use a proximity labelling-based proteomics strategy to map the interactome of the unique minus end-directed actin motor MYO6. Detailed biochemical and functional analysis identified several distinct MYO6-adaptor modules including two complexes containing RhoGEFs: the LIFT (LARG-Induced F-actin for Tethering) complex that controls endosome positioning and motility through RHO-driven actin polymerisation; and the DISP (DOCK7-Induced Septin disPlacement) complex, a novel regulator of the septin cytoskeleton. These complexes emphasise the role of MYO6 in coordinating endosome dynamics and cytoskeletal architecture. This study provides the first in vivo interactome of a myosin motor protein and highlights the power of this approach in uncovering dynamic and functionally diverse myosin motor complexes.

Filopodia formation and endosome clustering induced by mutant plus-end-directed myosin VI.

Myosin VI (MYO6) is the only myosin known to move toward the minus end of actin filaments. It has roles in numerous cellular processes, including maintenance of stereocilia structure, endocytosis, and autophagosome maturation. However, the functional necessity of minus-end-directed movement along actin is unclear as the underlying architecture of the local actin network is often unknown. To address this question, we engineered a mutant of MYO6, MYO6+, which undergoes plus-end-directed movement while retaining physiological cargo interactions in the tail. Expression of this mutant motor in HeLa cells led to a dramatic reorganization of cortical actin filaments and the formation of actin-rich filopodia. MYO6 is present on peripheral adaptor protein, phosphotyrosine interacting with PH domain and leucine zipper 1 (APPL1) signaling endosomes and MYO6+ expression causes a dramatic relocalization and clustering of this endocytic compartment in the cell cortex. MYO6+ and its adaptor GAIP interacting protein, C terminus (GIPC) accumulate at the tips of these filopodia, while APPL1 endosomes accumulate at the base. A combination of MYO6+ mutagenesis and siRNA-mediated depletion of MYO6 binding partners demonstrates that motor activity and binding to endosomal membranes mediated by GIPC and PI(4,5)P2 are crucial for filopodia formation. A similar reorganization of actin is induced by a constitutive dimer of MYO6+, indicating that multimerization of MYO6 on endosomes through binding to GIPC is required for this cellular activity and regulation of actin network structure. This unique engineered MYO6+ offers insights into both filopodia formation and MYO6 motor function at endosomes and at the plasma membrane.

Myosins: Domain Organisation, Motor Properties, Physiological Roles and Cellular Functions.

Myosins are cytoskeletal motor proteins that use energy derived from ATP hydrolysis to generate force and movement along actin filaments. Humans express 38 myosin genes belonging to 12 classes that participate in a diverse range of crucial activities, including muscle contraction, intracellular trafficking, cell division, motility, actin cytoskeletal organisation and cell signalling. Myosin malfunction has been implicated a variety of disorders including deafness, hypertrophic cardiomyopathy, Usher syndrome, Griscelli syndrome and cancer. In this chapter, we will first discuss the key structural and kinetic features that are conserved across the myosin family. Thereafter, we summarise for each member in turn its unique functional and structural adaptations, cellular roles and associated pathologies. Finally, we address the broad therapeutic potential for pharmacological interventions that target myosin family members.

Plasma membrane tension orchestrates membrane trafficking, cytoskeletal remodeling, and biochemical signaling during phagocytosis.

Phagocytes clear the body of undesirable particles such as infectious agents and debris. To extend pseudopods over the surface of targeted particles during engulfment, cells must change shape through extensive membrane and cytoskeleton remodeling. We observed that pseudopod extension occurred in two phases. In the first phase, pseudopods extended rapidly, with actin polymerization pushing the plasma membrane forward. The second phase occurred once the membrane area from preexisting reservoirs was depleted, leading to increased membrane tension. Increased tension directly altered the small Rho GTPase Rac1, 3'-phosphoinositide, and cytoskeletal organization. Furthermore, it activated exocytosis of vesicles containing GPI-anchored proteins, increasing membrane area and phagocytosis efficiency for large particles. We thus propose that, during phagocytosis, membrane remodeling, cytoskeletal organization, and biochemical signaling are orchestrated by the mechanical signal of membrane tension. These results put a simple mechanical signal at the heart of understanding immunological responses.

Restricted state selection in fluorescent protein Förster resonance energy transfer.

The measurement of donor lifetime modification by Förster resonance energy transfer (FRET) is a widely used tool for detecting protein-protein interactions and protein conformation change. Such measurements can be compromised by the presence of a significant noninteracting fraction of molecules. Combining time-resolved intensity and anisotropy measurements gives access to both molecular distance and orientation. Fluorescent proteins frequently used to detect energy transfer in biological systems often exhibit decay characteristics indicative of more than one excited state. However, little attention has thus far been given to the specific modes of energy transfer, in particular, which states are predominantly coupled. Here, we use a previously characterized dimerization system to study energy transfer between EGFP and mCherry. Optically excited EGFP and mCherry both exhibit biexponential decays, and FRET should therefore involve dipole-dipole transfer between these four states. Analysis of the sensitized fluorescence anisotropy and intensity decays indicates that FRET transfer is predominantly from the shorter lived EGFP emitting state (2.43 ns) to the longer lived (ca. 2.77 ns) minority component (ca. 16%) of the optically excited mCherry emission. This high degree of state selection between these two widely used FRET pairs highlights the fundamental differences that can arise between direct optical excitation of an isotropic molecular population and dipole-dipole coupling in a far from isotropic interaction geometry and has consequences regarding the accurate interpretation of fluorescent protein FRET data.

Myosin VI-Dependent Actin Cages Encapsulate Parkin-Positive Damaged Mitochondria.

Mitochondrial quality control is essential to maintain cellular homeostasis and is achieved by removing damaged, ubiquitinated mitochondria via Parkin-mediated mitophagy. Here, we demonstrate that MYO6 (myosin VI), a unique myosin that moves toward the minus end of actin filaments, forms a complex with Parkin and is selectively recruited to damaged mitochondria via its ubiquitin-binding domain. This myosin motor initiates the assembly of F-actin cages to encapsulate damaged mitochondria by forming a physical barrier that prevents refusion with neighboring populations. Loss of MYO6 results in an accumulation of mitophagosomes and an increase in mitochondrial mass. In addition, we observe downstream mitochondrial dysfunction manifesting as reduced respiratory capacity and decreased ability to rely on oxidative phosphorylation for energy production. Our work uncovers a crucial step in mitochondrial quality control: the formation of MYO6-dependent actin cages that ensure isolation of damaged mitochondria from the network.

MYO6 Regulates Spatial Organization of Signaling Endosomes Driving AKT Activation and Actin Dynamics.

APPL1- and RAB5-positive signaling endosomes play a crucial role in the activation of AKT in response to extracellular stimuli. Myosin VI (MYO6) and two of its cargo adaptor proteins, GIPC and TOM1/TOM1L2, localize to these peripheral endosomes and mediate endosome association with cortical actin filaments. Loss of MYO6 leads to the displacement of these endosomes from the cell cortex and accumulation in the perinuclear space. Depletion of this myosin not only affects endosome positioning, but also induces actin and lipid remodeling consistent with endosome maturation, including accumulation of F-actin and the endosomal lipid PI(3)P. These processes acutely perturb endosome function, as both AKT phosphorylation and RAC-dependent membrane ruffling were markedly reduced by depletion of either APPL1 or MYO6. These results place MYO6 and its binding partners at a central nexus in cellular signaling linking actin dynamics at the cell surface and endosomal signaling in the cell cortex.

F-actin waves, actin cortex disassembly and focal exocytosis driven by actin-phosphoinositide positive feedback.

Actin polymerization is controlled by the phosphoinositide composition of the plasma membrane. However, the molecular mechanisms underlying the spatiotemporal regulation of actin network organization over extended length scales are still unclear. To observe phosphoinositide-dependent cytoskeletal dynamics we combined the model system of frustrated phagocytosis, total internal reflection microscopy and manipulation of the buffer tonicity. We found that macrophages interacting with IgG-coated glass substrates formed circular F-actin waves on their ventral surface enclosing a region of plasma membrane devoid of cortical actin. Plasma membrane free of actin cortex was strongly depleted of PI(4,5)P2 , but enriched in PI(3,4)P2 and displayed a fivefold increase in exocytosis. Wave formation could be promoted by application of a hypotonic shock. The actin waves were characteristic of a bistable wavefront at the boundary between the regions of membrane containing and lacking cortical actin. Phosphoinositide modifiers and RhoGTPase activities dramatically redistributed with respect to the wavefronts, which often exhibited spatial oscillations. Perturbation of either lipid or actin cytoskeleton-related pathways led to rapid loss of both the polarized lipid distribution and the wavefront. As waves travelled over the plasma membrane, wavefront actin was seen to rapidly polymerize and depolymerize at pre-existing clusters of FcγRIIA, coincident with rapid changes in lipid composition. Thus the potential of receptors to support rapid F-actin polymerization appears to depend acutely on the local concentrations of multiple lipid species. We propose that interdependence through positive feedback from the cytoskeleton to lipid modifiers leads to coordinated local cortex remodeling, focal exocytosis, and organizes extended actin networks.

Mechanical feedback between membrane tension and dynamics.

The plasma membrane represents a physical inelastic barrier with a given area that adheres to the underlying cytoskeleton. The tension in the membrane physically affects cell functions and recent studies have highlighted that this physical signal orchestrates complex aspects of trafficking and motility. Despite its undeniable importance, little is known about the mechanisms by which membrane tension regulates cell functions or stimulates signals. The maintenance of membrane tension is also a matter of debate, particularly the nature of the membrane reservoir and trafficking pathways that buffer tension. In this review we discuss the importance of membrane area and of tension as a master integrator of cell functions, particularly for membrane traffic.

Regulation of 3-phosphoinositide-dependent protein kinase 1 activity by homodimerization in live cells.

3-Phosphoinositide-dependent kinase 1 (PDK1) plays a central role in regulating the activity of protein kinases that are essential for signaling; however, how PDK1 itself is regulated is largely unknown. We found that homodimerization of PDK1 is a spatially and temporally regulated mechanism for controlling PDK1 activity. We used Förster resonance energy transfer monitored by fluorescence lifetime imaging microscopy to observe PDK1 homodimerization in live cells. A pleckstrin homology (PH) domain-dependent, basal dimeric association of PDK1 was increased upon cell stimulation with growth factors; this association was prevented by a phosphatidylinositol 3-kinase inhibitor and by a mutation in, or a complete deletion of, the PH domain of PDK1. The distinct spatial distribution of PDK1 homodimers relative to that of heterodimers of PDK1 and protein kinase B (PKB), and the ability of monomeric mutants of PDK1 to phosphorylate PKB, suggested that the monomer was the active conformation. Mutation of the autophosphorylation residue threonine-513 to glutamate, which was predicted to destabilize the homodimer interface, enhanced the interaction between PDK1 and PKB and the activity of PKB. Through in vitro, time-resolved fluorescence intensity and anisotropy measurements, combined with existing crystal structures and computational molecular modeling, we determined the geometrical arrangement of the PDK1 homodimer. With this approach, we calculated the size of the population of PDK1 dimers in cells. This description of a previously uncharacterized regulatory mechanism for the activation of PDK1 offers possibilities for controlling PDK1 activity therapeutically.

Heredopathia atactica polyneuritiformis (Refsum's disease): 1. Clinical features and dietary management.

In a patient with Refsum's disease successful dietary control of the disease has been shown to depend on adequate energy and protein intake from phytanic acid-free sources and restriction of dietary phytanic acid to a maximum of 10 mg per day, with the provision of a generous amount of the essential fatty acid, linoleic acid. The present work gives data on the phytanic-acid content of various foodstuffs and suggests dietary manipulation of the patient. Although the role that chlorophyll-bound phytol plays in Refsum's disease is uncertain, it is advisable that this is eliminated until its role is more clearly identified.

Heredopathia atactica polyneuritiformis (Refsum's disease): 2. Estimation of phytanic acid in foods.

Estimation of phytanic acid in human plasma and in a number of foodstuffs is described.

Selective effect of low protein diets in chronic renal diseases.

It has recently been established that the rate of progression of chronic renal failure in man can be slowed by restricting dietary protein. Consequently, the short term and long term effects of a low protein diet on the course of different chronic nephropathies were studied in an attempt to delineate the factors that determine the response to such a diet. When a low protein diet was given for six months renal function improved significantly in nine patients with chronic tubulointerstitial nephritis (p less than 0.025); the diet had a marginally beneficial effect in 12 patients with chronic glomerulonephritis (p less than 0.05) and no effect in nine with hypertensive nephrosclerosis. The heterogeneous functional response in the patients with chronic glomerulonephritis correlated closely with the effect of the diet on these patients' proteinuria (r = 0.76, p less than 0.01). In a short term study (four weeks) of 12 patients with chronic renal failure changes in renal plasma flow were proportional to dietary protein intake. Renal vascular resistance fell during a high protein diet and increased when dietary protein was restricted. The changes in renal plasma flow during the low protein diet correlated well with the patients' long term functional response to the diet (r = 0.76, p less than 0.01). It is concluded that the response to a low protein diet in chronic renal failure is determined, firstly, by the nature of the underlying nephropathy, with maximal benefit being observed in non-glomerular disorders; secondly, by the effect of the diet on the proteinuria in chronic glomerulonephritis; and, thirdly, by the haemodynamic response to the diet, with patients with a reactive renal vascular bed improving with a low protein diet.