[Show abstract][Hide abstract] ABSTRACT: The actin nodule is a novel F-actin structure present in platelets during early spreading. However, only limited detail is known regarding nodule organization and function. Here we use electron microscopy, SIM and dSTORM super-resolution, and live-cell TIRF microscopy to characterize the structural organization and signalling pathways associated with nodule formation. Nodules are composed of up to four actin-rich structures linked together by actin bundles. They are enriched in the adhesion-related proteins talin and vinculin, have a central core of tyrosine phosphorylated proteins and are depleted of integrins at the plasma membrane. Nodule formation is dependent on Wiskott-Aldrich syndrome protein (WASp) and the ARP2/3 complex. WASp(-/-) mouse blood displays impaired platelet aggregate formation at arteriolar shear rates. We propose actin nodules are platelet podosome-related structures required for platelet-platelet interaction and their absence contributes to the bleeding diathesis of Wiskott-Aldrich syndrome.
Full-text · Article · Jun 2015 · Nature Communications
[Show abstract][Hide abstract] ABSTRACT: Total internal reflection fluorescence (TIRF) microscopy has gained popularity in recent years among cell biologists due to its ability to clearly visualize events that occur at the adherent plasma membrane of cells. TIRF microscopy systems are now commercially available from nearly all microscope suppliers. This review aims to give the reader an introduction to the physical basis of TIRF and considerations that need to be made when purchasing a commercial system. We explain how TIRF can be combined with other microscopy modalities and describe how to use TIRF to study processes such as endocytosis, exocytosis, and focal adhesion dynamics. Finally, we provide a step-by-step guide to imaging and analyzing focal adhesion dynamics in a migrating cell using TIRF microscopy.
No preview · Article · Jan 2015 · Methods in molecular biology (Clifton, N.J.)
[Show abstract][Hide abstract] ABSTRACT: Although fluorescence microscopy has had a major impact on biomedical research, the resolution barrier inherent in light microscopy restricts the ability to differentiate between objects closer together than ∼250 nm and prevents the true sizing of structures smaller than this limit. Recent innovations have led to the development of three main commercially available options for super-resolution microscopy that effectively break this diffraction limit: structured illumination microscopy (SIM), stochastic optical reconstruction microscopy (STORM)/photoactivation localization microscopy (PALM), and stimulated emission depletion microscopy (STED). The goal of this chapter is to describe the physical basis for these techniques as well as practical information for each, to provide the potential user with a basis for comparison and determination of the optimal choice for specific applications. Finally, innovative variations of these techniques for particular biological studies, as well as descriptions of new alternative techniques are presented.
[Show abstract][Hide abstract] ABSTRACT: Tight junctions (TJs) link adjacent cells and are critical for maintenance of apical-basolateral polarity in epithelial monolayers. The TJ protein occludin functions in disparate processes, including wound healing and Hepatitis C Virus infection. Little is known about steady-state occludin trafficking into and out of the plasma membrane. Therefore, we determined the mechanisms responsible for occludin turnover in confluent Madin-Darby canine kidney (MDCK) epithelial monolayers. Using various biotin-based trafficking assays we observed continuous and rapid endocytosis of plasma membrane localised occludin (the majority internalised within 30 minutes). By 120 minutes a significant reduction in internalised occludin was observed. Inhibition of lysosomal function attenuated the reduction in occludin signal post-endocytosis and promoted co-localisation with the late endocytic system. Using a similar method we demonstrated that ∼20% of internalised occludin was transported back to the cell surface. Consistent with these findings, significant co-localisation between internalised occludin and recycling endosomal compartments was observed. We then quantified the extent to which occludin synthesis and transport to the plasma membrane contributes to plasma membrane occludin homeostasis, identifying inhibition of protein synthesis led to decreased plasma membrane localised occludin. Significant co-localisation between occludin and the biosynthetic secretory pathway was demonstrated. Thus, under steady-state conditions occludin undergoes turnover via a continuous cycle of endocytosis, recycling and degradation, with degradation compensated for by biosynthetic exocytic trafficking. We developed a mathematical model to describe the endocytosis, recycling and degradation of occludin, utilising experimental data to provide quantitative estimates for the rates of these processes.
[Show abstract][Hide abstract] ABSTRACT: Previously we have shown that expression of the deubiquitinating enzyme USP17 is required for cell proliferation and motility. More recently we reported that USP17 deubiquitinates RCE1 isoform 2 and thus regulates the processing of 'CaaX' motif proteins. Here we now show that USP17 expression is induced by epidermal growth factor and that USP17 expression is required for clathrin mediated endocytosis of epidermal growth factor receptor. In addition, we show that USP17 is required for the endocytosis of transferrin, an archetypal substrate for clathrin mediated endocytosis, and that USP17 depletion impedes plasma membrane recruitment of the machinery required for clathrin mediated endocytosis. Thus, our data reveal that USP17 is necessary for epidermal growth factor receptor and transferrin endocytosis via clathrin coated pits, indicate this is mediated via the regulation of the recruitment of the components of the endocytosis machinery and suggest USP17 may play a general role in receptor endocytosis.
[Show abstract][Hide abstract] ABSTRACT: RhoJ is a RhoGTPase expressed in endothelial cells and tumour cells which regulates cell motility, invasion, endothelial tube formation and focal adhesion numbers. This study aimed to further delineate the molecular function of RhoJ. Using timelapse microscopy RhoJ was found to regulate focal adhesion disassembly; siRNA-mediated knockdown of RhoJ increased focal adhesion disassembly time, while expression of an active mutant (daRhoJ) decreased it. Further, daRhoJ co-precipitated with the GIT-PIX complex, a regulator of focal adhesion disassembly. An interaction between daRhoJ and GIT1 was confirmed using yeast-2-hybrid, which depended on the Spa homology domain of GIT1. GIT1, GIT2, β-PIX and RhoJ all co-localised in focal adhesions and depended on each other for their recruitment to focal adhesions. Functionally, the GIT-PIX complex regulated endothelial tube formation, with knockdown of GIT1/2 or β-PIX phenocopying RhoJ knockdown. RhoJ knockout mice showed reduced tumour growth and diminished tumour vessel density, identifying a role for RhoJ in mediating tumour angiogenesis. These studies give novel insight into the molecular function of RhoJ in regulating cell motility and tumour vessel formation.
Full-text · Article · Jun 2014 · Journal of Cell Science
[Show abstract][Hide abstract] ABSTRACT: Directed cell migration is critical for numerous physiological processes including development and wound healing. However chemotaxis is also exploited during cancer progression. Recent reports have suggested links between vesicle trafficking pathways and directed cell migration. Very little is known about the potential roles of endocytosis pathways during metastasis. Therefore we performed a series of studies employing a previously characterised model for chemotactic invasion of cancer cells to assess specific hypotheses potentially linking endocytosis to directed cell migration. Our results demonstrate that clathrin-mediated endocytosis is indispensable for epidermal growth factor (EGF) directed chemotactic invasion of MDA-MB-231 cells. Conversely, caveolar endocytosis is not required in this mode of migration. We further found that chemoattractant receptor (EGFR) trafficking occurs by clathrin-mediated endocytosis and is polarised towards the front of migrating cells. However, we found no role for clathrin-mediated endocytosis in focal adhesion disassembly in this migration model. Thus, this study has characterised the role of endocytosis during chemotactic invasion and has identified functions mechanistically linking clathrin-mediated endocytosis to directed cell motility.
[Show abstract][Hide abstract] ABSTRACT: Directed cell migration is critical for numerous physiological processes including development and wound healing. However chemotaxis is also exploited during cancer progression. Recent reports have suggested links between vesicle trafficking pathways and directed cell migration. Very little is known about the potential roles of endocytosis pathways during metastasis. Therefore we performed a series of studies employing a previously characterised model for chemotactic invasion of cancer cells to assess specific hypotheses potentially linking endocytosis to directed cell migration. Our results demonstrate that clathrin‐mediated endocytosis is indispensable for epidermal growth factor (EGF) directed chemotactic invasion of MDA‐MB‐231 cells. Conversely, caveolar endocytosis is not required in this mode of migration. We further found that chemoattractant receptor (EGFR) trafficking occurs by clathrin‐mediated endocytosis and is polarised towards the front of migrating cells. However, we found no role for clathrin‐mediated endocytosis in focal adhesion disassembly in this migration model. Thus, this study has characterised the role of endocytosis during chemotactic invasion and has identified functions mechanistically linking clathrin‐mediated endocytosis to directed cell motility.
[Show abstract][Hide abstract] ABSTRACT: Epidermal growth factor (EGF) receptor (EGFR) signalling regulates diverse cellular functions, promoting cell proliferation, differentiation, migration, cell growth and survival. EGFR signalling is critical during embryogenesis, in particular in epithelial development, and disruption of the EGFR gene results in epithelial immaturity and perinatal death. EGFR signalling also functions during wound healing responses through accelerating wound re-epithelialisation, inducing cell migration, proliferation and angiogenesis. Upregulation of EGFR signalling is often observed in carcinomas and has been shown to promote uncontrolled cell proliferation and metastasis. Therefore aberrant EGFR signalling is a common target for anticancer therapies. Various reports indicate that EGFR signalling primarily occurs at the plasma membrane and EGFR degradation following endocytosis greatly attenuates signalling. Other studies argue that EGFR internalisation is essential for complete activation of downstream signalling cascades and that endosomes can serve as signalling platforms. The aim of this review is to discuss current understanding of intersection between EGFR signalling and trafficking.
No preview · Article · Mar 2014 · The international journal of biochemistry & cell biology
[Show abstract][Hide abstract] ABSTRACT: Epithelial layers are integral for many physiological processes and are maintained by intercellular adhesive structures. During disease, these structures can disassemble, leading to breakdown of epithelia. TJs (tight junctions) are one type of intercellular adhesion. Loss of TJs has been linked to the pathogenesis of many diseases. The present review focuses on the role of vesicle trafficking in regulation of TJs, in particular trafficking of the TJ protein occludin. We examine how endocytosis and endosomal recycling modulate occludin localization under steady-state conditions and during stimulated TJ disassembly.
No preview · Article · Feb 2014 · Biochemical Society Transactions
[Show abstract][Hide abstract] ABSTRACT: Growth factor signalling regulates multiple cellular functions and its misregulation has been linked to cancer development and progression. Ack1 (Activated Cdc42-associated kinase 1, TNK2), a non-receptor tyrosine kinase, has been implicated in trafficking and degradation of epidermal growth factor receptor (EGFR), yet the precise functions remain elusive. In this report we investigate the role of Ack1 in EGFR trafficking and show that Ack1 partially colocalises to Atg16L-positive structures upon EGF stimulation. These are proposed to be the isolation membranes during autophagosome formation. In addition we find that Ack1 colocalises and interacts with sequestosome 1 (p62/SQSTM1), a receptor for selective autophagy, via a ubiquitin associated domain and this interaction decreases upon EGF treatment, thus suggesting that Ack1 moves away from p62/SQSTM1 compartments. Furthermore, Ack1 interacts and colocalises with NBR1, another autophagic receptor, and this colocalisation is enhanced in the presence of ectopically expressed p62/SQSTM1. Finally, Ack1 knock-down results in accelerated lysosomal localisation of EGFR upon EGF treatment. Structure-function analyses of a panel of Ack1 deletion mutants have revealed key mechanistic aspects of these relationships. The Mig6-homology domain and clathrin binding domain both contribute to the colocalisation with EGFR, whereas the UBA domain is critical for the colocalisation with p62/SQSTM1, but not NBR1. Taken together, our studies demonstrate a novel role for Ack1 in diverting activated EGFR into a non-canonical degradative pathway, marked by association with p62/SQSTM1, NBR1 and Atg16L.
No preview · Article · Jan 2014 · Journal of Cell Science
[Show abstract][Hide abstract] ABSTRACT: An understanding of the relationship between the physiochemical properties of nanoparticles and their uptake into cells is crucial for realising the potential of nanoparticles for biomedical applications and for developing strategies to minimise human and environmental nanotoxicity. In this work we studied the permeation of lipid bilayer membranes by uncharged hydrophobic polystyrene nanoparticles (PSNPs) as a function of nanoparticle size by means of coarse-grained molecular dynamics simulations. We also investigated the effect of changing the membrane environment by adding cholesterol to the bilayer. Free energy calculations reveal that overall it is energetically favourable for PSNPs to insert into the hydrocarbon interior of dipalmitoylphosphatidylcholine (DPPC) bilayers; however PSNPs with a diameter greater than the bilayer thickness are less readily accommodated than smaller PSNPs. As a PSNP approaches the bilayer there is an increase in the curvature of the bilayer as it bends and partially engulfs the PSNP. The energetic cost of bending is compensated for by the removal of unfavourable PSNP–water contacts. Despite cholesterol increasing the order and rigidity of the bilayer, the interior of the DPPC–cholesterol bilayer appears to be a more favourable environment for PSNPs, possibly due to stronger intermolecular interactions between polystyrene and the model cholesterol molecule than between polystyrene and DPPC.
[Show abstract][Hide abstract] ABSTRACT: Calcium phosphates are used widely as orthopaedic implants and in nanocrystalline form to enable the transfer of genetic material into cells. Despite widespread use, little is known about their fate after they have crossed the cell membrane. Here we present a method of surface modification of silicon-substituted hydroxyapatite (SiHA) through a silane group, which enables the engraftment of a fluorescent dye to facilitate real-time biological tracking. Surface modification of the nanocrystal surface was undertaken using (3-mercaptopropyl)trimethoxysilane (MPTS), which presented a thiol for the further attachment of a fluorophore. Successful modification of the surface was demonstrated using zeta potential measurements and fluorescence microscopy and the number of thiol groups at the surface was quantified using Ellman's reagent. In vitro experiments using the fluorescently modified particles enabled the discrimination of the calcium phosphate particulate from other biological debris following internalisation by a population of MC3T3 (pre-osteoblast) cells and the particles were shown to maintain fluorescence for 24 hours without quenching. The successful modification of the surface of SiHA with thiol groups offers the tantalising possibility of the intracellular growth factor delivery.
[Show abstract][Hide abstract] ABSTRACT: Eps8 is involved in both cell signalling and receptor trafficking. It is a known phosphorylation substrate for two proteins involved in the fibroblast growth factor receptor (FGFR) signalling pathway: the receptor itself and Src. Here we report a differential proteomic analysis of Eps8 aimed to identify specific FGFR and Src family kinase dependent phosphosites and co-associated phosphodependent binding partners. This study reveals a total of 22 Eps8 pTyr and pSer/Thr phosphorylation sites, including those that are dependent on Src family and FGFR kinase activity. Peptide affinity purification of proteins that bind to a selection of the pTyr phosphosites has identified a range of novel Eps8 binding partners including members of the intracellular vesicle trafficking machinery (clathrin and AP-2), proteins which have been shown to regulate activated receptor trafficking (NBR1 and Vav2), and proteins involved in receptor signalling (IRS4 and Shp2). Collectively this study significantly extends the understanding of Eps8 post-translational modification by regulated phosphorylation, identifies novel Eps8 binding partners implicated in receptor trafficking and signalling, and confirms the functions of Eps8 at the nexus of receptor signalling and vesicular trafficking.
[Show abstract][Hide abstract] ABSTRACT: Fibroblast Growth Factor Receptors (FGFRs) mediate a wide spectrum of cellular responses critical for development and wound healing. However, aberrant FGFR activity leads to cancer. Activated growth factor receptors undergo stimulated endocytosis, but can continue to signal along the endocytic pathway. Endocytic trafficking controls the duration and intensity of signalling, and growth factor receptor signalling can lead to modifications of trafficking pathways. We have developed live-cell imaging methods for studying FGFR dynamics to investigate mechanisms that coordinate the interplay between receptor trafficking and signal transduction. Activated FGFR enters the cell following recruitment to pre-formed clathrin-coated pits (CCPs). However, FGFR activation stimulates clathrin-mediated endocytosis; FGF treatment increases the number of CCPs, including those undergoing endocytosis, and this effect is mediated by Src and its phosphorylation target Eps8. Eps8 interacts with the clathrin-mediated endocytosis machinery and depletion of Eps8 inhibits FGFR trafficking and immediate Erk signalling. Once internalized, FGFR passes through peripheral early endosomes en route to recycling and degredative compartments, via a Src and Eps8 dependent mechanism. Thus Eps8 functions as a key coordinator in the interplay between FGFR signalling and trafficking. This work provides the first detailed mechanistic analysis from growth factor receptor clustering at the cell surface to signal transduction and endocytic trafficking. As we have characterised the Src target Eps8 as a key regulator of FGFR signalling and trafficking, and identified the early endocytic system as the site of Eps8 mediated effects, this work provides novel mechanistic insight into the reciprocal regulation of growth factor receptor signalling and trafficking.
Full-text · Article · Nov 2012 · Journal of Cell Science