Figure 3 - uploaded by George Galea
Content may be subject to copyright.
Primary RNAi screen of the Rab library for NP delivery to LAMP1-positive membranes and validation of candidates. (a) Graph showing LAMP1-associated NP ratio in cells treated with a library of siRNAs targeting 58 RAB genes. (b) Example confocal images of HeLa cells transfected with non-targeting siRNA (siNEG) or targeting RAB7A or RAB33B and then pulse-chased with 40 nm PS-COOH NPs for 60 minutes, fixed and immunostained for LAMP1. In the merged images, nuclei are in blue, LAMP1 in green and NPs in red. Arrowheads indicate co-localizing structures. Scale bars, 20 μ m and 6 μ m in zoom. (c,e,f) Graphs showing percentage of NPs co-localizing with LAMP1-positive membranes in control cells (NEG) or cells treated with siRNAs targeting RAB7A, RAB33B, DNM2 or combinations as indicated. (d) Graph showing the percentage of NPs co-localizing with LAMP1-positive membranes in control cells (NEG), or treated with RAB33B siRNA and then either left untreated (siRAB33B) or transfected with an siRNA-resistant GFP-RAB33B construct (Rab33b* ). In (a) values represent mean and s.e.m. of 3 independent experiments normalized to the value shown by cells treated with siNEG. In (c–f) values represent mean and s.e.m. of 3 independent experiments; * p-value < 0.05; * * * p-value < 0.001.
Source publication
Synthetic nanoparticles are promising tools for imaging and drug delivery; however the molecular details of cellular internalization and trafficking await full characterization. Current knowledge suggests that following endocytosis most nanoparticles pass from endosomes to lysosomes. In order to design effective drug delivery strategies that can us...
Contexts in source publication
Context 1
... further validation, selected members of each family identified in the siRNA screen were analyzed by qPCR ( Figure S3 in Supporting Information). Together these experiments revealed, for the first time, sets of proteins influencing NP uptake and trafficking. ...
Context 2
... of organelle-specificity and we have previously used live-cell imaging to show that PS-COOH NPs pass through membranes decorated with Rab5, Rab9 and Rab7 30 . Of the 58 Rabs targeted in the RNAi approach described here, depletion of 11 of them resulted in a mild decrease of LAMP1-positive organelle delivery, while 4 displayed a strong decrease ( Fig. 3a and Supporting Information Table ...
Context 3
... effects observed on depletion of RAB7A and RAB33B. We repeated our NP uptake experiments in cells depleted for each of these mRNA targets, now using confocal images, and carried out precise quantitative co-localization analysis using the Rank Weighted Co-localization (RWC) coefficient 31 as a measure of the co-occurrence of NP signal and LAMP1 (Fig. 3b). The measurements obtained were similar to those seen in the automated analysis, clearly showing that NP delivery to lysosomes is affected by the levels of these two proteins (Fig. 3c). Western blot analysis confirmed the successful depletion of Rab33b protein in cells treated with siRNAs targeting RAB33B mRNA (Supporting Information ...
Context 4
... precise quantitative co-localization analysis using the Rank Weighted Co-localization (RWC) coefficient 31 as a measure of the co-occurrence of NP signal and LAMP1 (Fig. 3b). The measurements obtained were similar to those seen in the automated analysis, clearly showing that NP delivery to lysosomes is affected by the levels of these two proteins (Fig. 3c). Western blot analysis confirmed the successful depletion of Rab33b protein in cells treated with siRNAs targeting RAB33B mRNA (Supporting Information Figure S6). Visual examination of a number of the images revealed occasional changes in cellular distribution of the lysosome population. Therefore, to confirm that any significant ...
Context 5
... would make the mRNA resistant to RAB33B siRNA treatment. Overexpression of this construct in cells also incubated with the siRNAs targeting RAB33B was able to partially recover the levels of co-localization of NPs with LAMP1 to that seen in control cells, strongly supporting the notion that Rab33b is a direct regulator of NP delivery to lysosomes (Fig. 3d). Interestingly, the combined deple- tion of DNM2 with either RAB7A or RAB33B in both cases resulted in an additive (although not statistically significant) effect in terms of a further reduction of delivery of NPs to LAMP1-positive organelles (Fig. 3c,e,f), consistent with distinct roles for DNM2 at the cell surface and the two Rabs on ...
Context 6
... strongly supporting the notion that Rab33b is a direct regulator of NP delivery to lysosomes (Fig. 3d). Interestingly, the combined deple- tion of DNM2 with either RAB7A or RAB33B in both cases resulted in an additive (although not statistically significant) effect in terms of a further reduction of delivery of NPs to LAMP1-positive organelles (Fig. 3c,e,f), consistent with distinct roles for DNM2 at the cell surface and the two Rabs on internal membranes. Since these results supporting a role for the late endosomal Rab7a in NP trafficking were in good agreement with our previ- ous observations 30 , we decided to further investigate Rab33b, as this has never previously been linked to NP ...
Similar publications
Degradation of maternal proteins by the ubiquitin-proteasome system (UPS) accompanies the maternal-to-zygotic transition. DPPA3/Stella/PGC7, encoded by a maternal effect gene, is present in the nucleus and cytoplasm of zygotes and has been associated with protecting the female pronucleus from TET3-mediated demethylation. We now report that cytoplas...
Annexin A1 (ANXA1) is an endogenous protein with potent anti-inflammatory properties in the brain. Although ANXA1 has been predominantly studied for its binding to formyl peptide receptors (FPRs) on plasma membranes, little is known regarding whether this protein has an anti-inflammatory effect in the cytosol. Here, we investigated the mechanism by...
Introduction
Cell biological and genetic evidence implicates failures in degrading aggregating proteins, such as tau and TDP‐43, through autophagy or lysosomal pathways in the pathogenesis of Frontotemporal Lobar degeneration (FTLD).
Methods
We investigated changes in degradative pathways in 60 patients with different pathological or genetic forms...
Background
Flow cytometric platelet activation has emerged as an alternative diagnostic test for inherited platelet disorders. It is, however, labor intensive and few studies have directly compared the performance of flow cytometric platelet activation (PACT) to light transmission aggregometry (LTA). The aims of this study were 1/ to develop a simp...
IκB, a cytoplasmic inhibitor of nuclear factor-κB (NF-κB), is reportedly degraded via the proteasome. However, we recently found that long-term incubation with proteasome inhibitors (PIs) such as PS-341 or MG132 induces IκBα degradation via an alternative pathway, lysosome, which results in NF-κB activation and confers resistance to PI-induced lung...
Citations
... commonly applied in many nanomedicine and endocytosis studies. 12,14,37,38 Confocal fluorescence imaging confirmed nanoparticle uptake and accumulation in the lysosomes ( Figure S2a). Uptake kinetics showed up to 70% uptake reduction in LDLR silenced cells, the effect being stronger at longer exposure times ( Figure S2b). ...
Nanosized drug carriers enter cells via active mechanisms of endocytosis but the pathways involved are often not clarified. Cells possess several mechanisms to generate membrane curvature during uptake. However, the mechanisms of membrane curvature generation for nanoparticle uptake have not been explored so far. Here, we combined different methods to characterize how silica nanoparticles with a human serum corona enter cells. In these conditions, silica nanoparticles are internalized via the LDL receptor (LDLR). We demonstrate that despite the interaction with LDLR, uptake is not clathrin-mediated, as usually observed for this receptor. Additionally, silencing the expression of different proteins involved in clathrin-independent mechanisms and several BAR-domain proteins known to generate membrane curvature strongly reduces nanoparticle uptake. Thus, nanosized objects targeted to specific receptors, such as here LDLR, can enter cells via different mechanisms than their endogenous ligands. Additionally, nanoparticles may trigger alternative mechanisms of membrane curvature generation for their internalization.
... RNAi screening of approximately 400 genes associated with membrane traffic and cytoskeleton regulation was initially used to define the key machinery associated with delivery of polymeric nanoparticles to lysosomes. Much of this work was also focused on the Rab GTPases and identified Rab33b as being a critical regulator of nanoparticle trafficking to lysosomes in monolayer cells [37]. Subsequently, we repeated this RNAi approach, albeit on a pilot scale, but using a solid spheroid model of HT-29 adenocarcinoma cells [38]. ...
The endomembrane system of mammalian cells provides massive capacity for the segregation of biochemical reactions into discrete locations. The individual organelles of the endomembrane system also require the ability to precisely transport material between these compartments in order to maintain cell homeostasis; this process is termed membrane traffic. For several decades, researchers have been systematically identifying and dissecting the molecular machinery that governs membrane trafficking pathways, with the overwhelming majority of these studies being carried out in cultured cells growing as monolayers. In recent years, a number of methodological innovations have provided the opportunity for cultured cells to be grown as 3-dimensional (3D) assemblies, for example as spheroids and organoids. These structures have the potential to better replicate the cellular environment found in tissues and present an exciting new opportunity for the study of cell function. In this mini-review, we summarize the main methods used to generate 3D cell models and highlight emerging studies that have started to use these models to study basic cellular processes. We also describe a number of pieces of work that potentially provide the basis for adaptation for deeper study of how membrane traffic is coordinated in multicellular assemblies. Finally, we comment on some of the technological challenges that still need to be overcome if 3D cell biology is to become a mainstream tool toward deepening our understanding of the endomembrane system in mammalian cells.
... RNAi screening of approximately 400 genes associated with membrane traffic and cytoskeleton regulation was initially used to define the key machinery associated with delivery of polymeric nanoparticles to lysosomes. Much of this work was also focussed on the Rab GTPases, and identified Rab33b as being a critical regulator of nanoparticle trafficking to lysosomes in monolayer cells [37]. Subsequently, we repeated this RNAi approach, albeit on a pilot scale, but using a solid spheroid model of HT-29 adenocarcinoma cells [38]. ...
The endomembrane system of mammalian cells provides massive capacity for the segregation of biochemical reactions into discrete locations. The individual organelles of the endomembrane system also require the ability to precisely transport material between these compartments in order to maintain cell homeostasis; this process is termed membrane traffic. For several decades, researchers have been systematically identifying and dissecting the molecular machinery that governs membrane trafficking pathways, with the overwhelming majority of these studies being carried out in cultured cells growing as monolayers. In recent years, a number of methodological innovations have provided the opportunity for cultured cells to be grown as 3-dimensional (3D) assemblies, for example as spheroids and organoids. These structures have the potential to better replicate the cellular environment found in tissues, and present an exciting new opportunity for the study of cell function. In this mini-review we summarise the main methods used to generate 3D cell models, and highlight emerging studies that have started to use these models to study basic cellular processes. We also describe a number of pieces of work that potentially provide the basis for adaptation for deeper study of how membrane traffic is coordinated in multicellular assemblies. Finally, we comment on some of the technological challenges that still need to be overcome if 3D cell biology is to become a mainstream tool towards deepening our understanding of the endomembrane system in mammalian cells.
... Hence, such genetic perturbation technologies can also be used to gain understanding on or modulate the function of specific genes involved in functional nucleic acid delivery. However, to date only smallscale studies (either through selected perturbation of a limited number of genes [179,180,201,[213][214][215][216][217][218][219][220][221] or relatively small targeted screens [222][223][224][225][226]) have investigated the genetic basis of endocytosis [179,201,215], intracellular trafficking [213,225], cytotoxicity [214] or effective delivery [180,[215][216][217][218][219][220][221][222][223][224]226] of nucleic acids or nucleic acid-loaded nanoparticles [227]. Both RNAi and CRISPR/Cas9 technology, the latter which allows generation of gene knockouts (CRISPRko), or transcriptional upregulation (CRISPR activation; CRISPRa) or downregulation (CRISPR interference; CRISPRi) of genes, have an advantage over small molecules [228,229]. ...
... Hence, such genetic perturbation technologies can also be used to gain understanding on or modulate the function of specific genes involved in functional nucleic acid delivery. However, to date only smallscale studies (either through selected perturbation of a limited number of genes [179,180,201,[213][214][215][216][217][218][219][220][221] or relatively small targeted screens [222][223][224][225][226]) have investigated the genetic basis of endocytosis [179,201,215], intracellular trafficking [213,225], cytotoxicity [214] or effective delivery [180,[215][216][217][218][219][220][221][222][223][224]226] of nucleic acids or nucleic acid-loaded nanoparticles [227]. Both RNAi and CRISPR/Cas9 technology, the latter which allows generation of gene knockouts (CRISPRko), or transcriptional upregulation (CRISPR activation; CRISPRa) or downregulation (CRISPR interference; CRISPRi) of genes, have an advantage over small molecules [228,229]. ...
RNA therapeutics (e.g. siRNA, oligonucleotides, mRNA, etc.) show great potential for the treatment of a myriad of diseases. However, to reach their site of action in the cytosol or nucleus of target cells, multiple intra- and extracellular barriers have to be surmounted. Several non-viral delivery systems, such as nanoparticles and conjugates, have been successfully developed to meet this requirement. Unfortunately, despite these clear advances, state-of-the-art delivery agents still suffer from relatively low intracellular delivery efficiencies. Notably, our current understanding of the intracellular delivery process is largely oversimplified. Gaining mechanistic insight into how RNA formulations are processed by cells will fuel rational design of the next generation of delivery carriers. In addition, identifying which intracellular pathways contribute to productive RNA delivery could provide opportunities to boost the delivery performance of existing nanoformulations. In this review, we discuss both established as well as emerging techniques that can be used to assess the impact of different intracellular barriers on RNA transfection performance. Next, we highlight how several modulators, including small molecules but also genetic perturbation technologies, can boost RNA delivery by intervening at differing stages of the intracellular delivery process, such as cellular uptake, intracellular trafficking, endosomal escape, autophagy and exocytosis.
... Interestingly, Rab3A is also found to specifically bind to wildtype GTP-locked Rab8A, which could be explained by both Rab8A and Rab3A being known to interact with SYTL4 (Fukuda, 2003). There are also a number of cytoskeletal/trafficking proteins enriched for the phospho-mutants, such as MON2, DCTN1/Dynactin subunit 1, and the WASH complex subunit WASHC2A for the non-phosphorylated (T72A) mutant (Fig. 4.1B) and MIS18A, TBC1D25/OATL1, and ARFGEF3/BIG3 for the phospho-mimicking mutant (T72E) (Fig. 4.1C) (Carter et al., 2016;Tao Liu et al., 2016;Mahajan et al., 2019;Panarella et al., 2016;Spiller et al., 2017;Jing Wang et al., 2018). In addition, the golgin GCC1/GCC88 appears to bind to the wildtype and the phosphomimicked mutant, but not to the non-phosphorylated mutant, suggesting that GCC88 requires phosphorylation (or the possibility of phosphorylation) to bind. ...
Secretion and endocytosis are essential processes in eukaryotic cells, executed and tightly regulated by the cell’s endomembrane system. It is widely accepted that small GTPases of the Ras superfamily are major regulators of membrane trafficking and signalling, ensuring specificity and efficiency through their spatiotemporal regulation.Identifying the proteins interacting with small GTPases is vital for understanding how the small G proteins are regulated and where they function. With the aim of eliminating certain limitations posed by many traditional methods of studying protein-protein interactions, an adaptation of the recently developed in vivo proximity labelling technique BioID was used in this study. Here, BioID was redirected to the ectopic location of the surface of mitochondria (hereafter called MitoID) with the aim of restricting the non-specific background. Applying this method to 25 small GTPases of the Ras superfamily in their active and inactive states allowed for a direct and clean comparison between the putative interactomes, identifying both known effectors and regulators as well as putative novel interactors for most tested GTPases. Several potential novel interactions were validated through GST affinity chromatography and/or microscopy, namely with the Rab GTPases Rab2A (ARFGEF3/BIG3, STAMBPL1), Rab5A (OSBPL9, TBCK), Rab9A (HPS3, NDE1), and Rab11A/B (ALS2). Furthermore, numerous novel interactors were identified for Rab1A and Rab1B, two mammalian paralogues of the yeast protein Ypt1 that are known to be key regulators in ER to Golgi trafficking and suggested to play a role in the regulation of autophagy. The newly identified proteins include Rabaptin5, which is a key player in the regulation of endosomal trafficking, PPP1R37, a protein of unknown function, CALCOCO1 and CLEC16A, which are potentially involved in autophagy, and several components of the phosphatidylinsositol-3-phospate kinase (PI3K) complex I, which is known to be a major autophagic regulator. In collaboration with the Williams group, the PI3K complex I was shown to be a bona fide Rab1A regulator. Furthermore, direct binding assays showed that CALCOCO1, CLEC16A, and PPP1R37 all bind to Rab1A and Rab1B independently of adaptor proteins or other factors. More specifically, the Rab1 binding site on PPP1R37 was shown to be located on the C-terminal and central regions that contain multiple leucine rich repeats. Collectively, this study has shown that MitoID is an effective and powerful tool to study protein-protein interactions, and has identified and confirmed several novel interactors of small GTPases which can aid in gaining understanding of how major processes such as vesicle trafficking and autophagy are regulated.
... The link between Rab33b and OATL1 has been further supported by evidence that both of these proteins are required for the uptake and transfer of synthetic nanoparticles to lysosomal compartments. RNAi-induced depletion of Rab33b reduces the accumulation of nanoparticles in lysosomes, but this can be rescued by the overexpression of wild-type Rab33b protein [93]. Additionally, overexpression of OATL1 was also found to reduce nanoparticle accumulation in acidic compartments [93]. ...
... RNAi-induced depletion of Rab33b reduces the accumulation of nanoparticles in lysosomes, but this can be rescued by the overexpression of wild-type Rab33b protein [93]. Additionally, overexpression of OATL1 was also found to reduce nanoparticle accumulation in acidic compartments [93]. Taken together, overexpression of OATL1 reduces the delivery of nanoparticles to lysosomal compartments, as well as the fusion of autophagosomes with lysosomes, both of which are processes that require Rab33b. ...
Autophagy (particularly macroautophagy) is a bulk degradation process used by eukaryotic cells in order to maintain adequate energy levels and cellular homeostasis through the delivery of long-lived proteins and organelles to the lysosome, resulting in their degradation. It is becoming increasingly clear that many of the molecular requirements to fulfil autophagy intersect with those of conventional and unconventional membrane trafficking pathways. Of particular interest is the dependence of these processes on multiple members of the Rab family of small GTP binding proteins. Rab33b is a protein that localises to the Golgi apparatus and has suggested functions in both membrane trafficking and autophagic processes. Interestingly, mutations in the RAB33B gene have been reported to cause the severe skeletal disorder, Smith–McCort Dysplasia; however, the molecular basis for Rab33b in this disorder remains to be determined. In this review, we focus on the current knowledge of the participation of Rab33b and its interacting partners in membrane trafficking and macroautophagy, and speculate on how its function, and dysfunction, may contribute to human disease.
... In order to optimize the use of this kind of compound and avoid misinterpretations, two requirements are essential: first, testing their efficacy in blocking the pathway of interest and second, testing their toxicity on the cells chosen for the study. To this aim, here, HeLa epithelial ovarian cancer cells were selected as a model cell line commonly used both in the endocytosis and in the nanomedicine fields [11,14,50,51]. ...
Aim: In this work we illustrate limits and challenges associated with the use of pharmacological inhibitors to study how nanomedicines enter cells and show how such limits can be overcome. Materials & methods: We selected a panel of six common pharmacological inhibitors and a model nanoparticle-cell system. We tested eventual toxicity by measuring cell viability. We confirmed drug efficacy by measuring the uptake of control markers for the pathways involved by flow cytometry and fluorescence microscopy. Results & conclusion: We show how to optimize the use of pharmacological inhibitors and interpret the results generated. Furthermore, we demonstrate that some inhibitors cannot be used for nanomedicine studies because they lose their efficacy when serum is added, as required for nanoparticle exposure to cells.
... NA LUCPLFLN objective lens. Image processing was performed using a Columbus Image Data Storage and Analysis System v2.3.1 (PerkinElmer) as previously described by Panarella et al. 29 Briefly, the Hoechst3334 channel was used to detect the cell nucleus and cytoplasm. Nanomedicine spots associated with each cell were then detected in the Cy3 channel and counted. ...
The field of tissue engineering is increasingly recognizing that gene therapy can be employed for modulating in vivo cellular response thereby guiding tissue regeneration. However, the field lacks a versatile and biocompatible gene delivery platform capable of efficiently delivering transgenes to mesenchymal stem cells (MSCs), a cell type refractory to transfection. Herein, we describe the extensive and systematic exploration of three architectural variations of star-shaped poly(L-lysine) polypeptide (star-PLL) with varying number and length of poly(L-Lysine) arms as potential non-viral gene delivery vectors for MSCs. We demonstrate that the star-PLL architecture is capable of self-assembling with pDNA to form stable, cationic nanomedicines. Utilizing high content screening, live cell imaging and mechanistic uptake studies we confirm the intracellular delivery of pDNA by star-PLLs is a rapid process which likely proceeds via a clathrin independent mechanism. We identify a star-PLL composition with 64 poly(L-Lysine) arms and 5 L-Lysine subunits per arm as a particularly efficient architecture which is capable of delivering both reporter genes and the therapeutic transgenes bone morphogenetic protein-2 and vascular endothelial growth factor to MSCs. This composition facilitated a 1000-fold increase in transgene expression in MSCs compared to its linear analogue, linear poly(L-Lysine). Furthermore, it demonstrated comparable transgene expression to widely used vector polyethylenimine using a lower pDNA dose with significantly less cytotoxicity. Overall, this study illustrates the ability of the star-PLL polymeric architecture to facilitate efficient, non-toxic nucleic acid delivery to MSCs thereby functioning as an innovative nanomedicine platform for tissue engineering applications.
... Our lack of understanding with their intracellular fate demands the development of strategies or aspects to take into account that overcomes the hurdles presented by them. Pranella et al. determined the key roles of proteins majorly responsible for the trafficking of polystyrene NPs into the HeLa cells by gene-based HCS i.e. combination of RNAi technology with automated HCS microscopy [96]. ...
