Biochimica et Biophysica Acta (BBA) - General Subjects
Missing-in-metastasis protein promotes internalization of magnetic nanoparticles via association with clathrin light chain and Rab7
Graphical abstract
Introduction
Magnetic nanoparticles (MNPs) possess unique physical and chemical properties [1] and have been extensively used in a variety of biomedical applications, including magnetic resonance imaging [2], heating mediators for hyperthermia [3], cell labeling and tracking [4], cell sensing [5], drug delivery [6] and theragnostics [7]. Many of these applications require the injection of nanomaterials into the blood circulation, and the success of their usages depends largely on the duration of the particles once internalized into targeted cells. Yet, the clearance profile of these nanomaterials within cells is unknown [8]. After entering tissues through the bloodstream, nanoparticles tend to complex with certain serum proteins, which forms a “corona” that influences greatly the biological property of the particles [[9], [10], [11]]. These serum proteins act as opsonin and facilitate the uptake of nanoparticles by the mononuclear phagocyte system (MPS), which is regarded as a limit step for long-circulating and targeting [[12], [13], [14]]. Therefore, minimizing the clearance of MPS, especially by macrophages, would likely increase their duration in vivo, thereby improving the efficacy of their applications [15,16].
A few endocytic pathways have been implicated in the uptake of nanoparticles into cells [17]. These include, but not limited to, clathrin/caveolae-mediated endocytosis, phagocytosis, macropinocytosis and pinocytosis. Among them, the clathrin-mediated endocytosis is considered as the major route for MNPs to be internalized into macrophages [18]. The entire process of clathrin-mediated endocytosis involves extensive membrane deformations, which are initiated by a series of membrane sculfing proteins. Many of these proteins belong to the Bin/amphiphysin/Rvs (BAR) domain superfamily, which includes several subfamilies, including BAR/N-BAR, EFC/F-BAR and I-BAR domain proteins [19]. These BAR domain proteins sense and generate membrane curvatures by binding or insertion into specific phospholipid membranes through a curved interface organized by their BAR domains [20]. BAR domain proteins that have been involved in the clathrin-mediated endocytosis include endophilin, amphiphysin, SNX9, FBP17 and syndapin [21]. In addition, FCHo1/Syp1 F-BAR proteins have also been implicated in the budding of the clathrin-coated pits. During the formation of clathrin-coated pits, amphiphysin and endophilin recruit dynamin to the neck region via their SH3 domains [22]. SNX9 contains several binding sites for AP-2 and localizes clathrin subunits to a budding neck, which ensures the generation of clathrin-coated vesicles [21,23].
Unlike traditional BAR domain proteins, the role of I-BAR domain proteins in the clathrin-mediated endocytosis is less understood. A representative of the I-BAR domain protein subfamily is MIM, or metastasis suppressor 1 (MTSS1), which regulates actin dynamics and promotes membrane deformation [19,24]. Although MIM does not have an SH3 domain, it interacts with multiple cellular proteins, including PTPδ, cortactin, Rac and AIP4, and has been involved in filopodia-like protrusive extensions, cell migration, cell-cell interaction and endocytosis [25]. Recent studies have also suggested that MIM participates in the trafficking of internalized cargos within cells [26,27]. However, the role of MIM in endocytosis is uncertain as drosophila MIM actually inhibits the endocytosis of EGFR by competing with endophilins [28], indicating that MIM may play different roles in endocytosis in a context-dependent manner.
We have recently observed that mammalian MIM is an effector of Rab GTPases in the endocytosis mediated by chemokine receptor CXCR4 and associated with late endosomes via binding to Rab7 [29], a small GTPase that plays a vital role in the maturation of late endosomes from early endosomes. Also, MIM is the only member of the I-BAR domain family that is highly expressed in macrophages [30] and plays a positive role in the internalization of nanoparticles [31]. However, it was unclear about specific process of MNPs in macrophages that is regulated by MIM. In the presented study, we attempted to investigate the MIM-mediated molecular events during the internalization of MNPs in macrophages and found that MNPs triggered the association of MIM with clathrin light chain (CLC) and Rab7. Interestingly, MIM failed to interact with clathrin heavy chain during early responses to MNPs. As both CLC and Rab7 are implicated in sorting of endocytic vesicles, our data suggest that MNPs are processed within macrophages through an intracellular traffic machinery involving the functions of MIM, CLC and Rab7.
Section snippets
Material and reagents
Lipofectamine 2000 transfection reagent (Cat. No. 11668027), anti-GFP antibody (Cat. No. A11122), FITC-conjugated goat anti-rabbit (Cat. No. A16097), Alexa flour 488-conjugated goat anti-rabbit IgG (Cat. No.A11008), and Alexa flour 568-conjugted goat anti-mouse IgG (Cat. No.A11004) antibodies were purchased from Invitrogen. Antibody against MIM (Cat. No. PA5-17047), Cell Dissociation Buffer (Cat. No. 13151014), protein A/G agarose beads (Cat. No. 20423) and G418 sulfate (Cat. No. 10131035) were
Characterization of Ferumoxytol nanoparticles
Ferumoxytol magnetic nanoparticles (MNPs),which were composed of an iron oxide core and a polyglucose sorbitol carboxymethyether (PSC) shell (Fig. 1A), were prepared as described previously [36]. The physical property of the prepared MNPs was initially examined by TEM, which revealed that most of the particles were quasi-spherical with an average diameter of 7 nm (Fig. 1B). Consistently, the average hydrodynamic size of MNPs was about 30.15 nm with an apparent zeta potential of −28.7 mV as
Conclusions
In summary, we have provided evidence for the role of MIM in the intracellular trafficking of MNPs via interactions with CLC, Rab5 and Rab7. This finding is novel because this is the first report about the interaction between MIM and CLC in response to extracellular stimuli. Yet, at an early phase of internalization we did not find significant interaction between MIM CHC, which is known to be assembled into clathrin lattices in the initial uptake of cargos during receptor-mediated endocytosis.
Acknowledgements
This work was supported by NSFC Projects of International Cooperation and Exchanges No. (61420106012), the National Key Research and Development Program of China (No. 2017YFA0104302), Collaborative Innovation Center of Suzhou Nano Science and Technology, the Fundamental Research Funds for the Central Universities (No. 2242017 K40232). This research was also supported by National Cancer Institute (R01 CA113809 to X. Z.). We thank Dr. Hongyin Wang from the University of Texas Health Science
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