Mechanisms Of Intracellular Trafficking Research Articles

Myosin XI, a model of its conserved role in plant cell tip growth.

In eukaryotic cells, organelle and vesicle transport, positioning, and interactions play crucial roles in cytoplasmic organization and function. These processes are governed by intracellular trafficking mechanisms. At the core of that trafficking, the cytoskeleton and directional transport by motor proteins stand out as its key regulators. Plant cell tip growth is a well-studied example of cytoplasm organization by polarization. This polarization, essential for the cell's function, is driven by the cytoskeleton and its associated motors. This review will focus on myosin XI, a molecular motor critical for vesicle trafficking and polarized plant cell growth. We will center our discussion on recent data from the moss Physcomitrium patens and the liverwort Marchantia polymorpha. The biochemical properties and structure of myosin XI in various plant species are discussed, highlighting functional conservation across species. We further explore this conservation of myosin XI function in the process of vesicle transport in tip-growing cells. Existing evidence indicates that myosin XI actively organizes actin filaments in tip-growing cells by a mechanism based on vesicle clustering at their tips. A hypothetical model is presented to explain the essential function of myosin XI in polarized plant cell growth based on vesicle clustering at the tip. The review also provides insight into the in vivo localization and dynamics of myosin XI, emphasizing its role in cytosolic calcium regulation, which influences the polymerization of F-actin. Lastly, we touch upon the need for additional research to elucidate the regulation of myosin function.

Rab37 mediates trafficking and membrane presentation of PD-1 to sustain T cell exhaustion in lung cancer

BackgroundProgrammed cell death protein 1 (PD-1) is an immune checkpoint receptor expressed on the surface of T cells. High expression of PD-1 leads to T-cell dysfunction in the tumor microenvironment (TME). However, the mechanism of intracellular trafficking and plasma membrane presentation of PD-1 remains unclear.MethodsMultiple databases of lung cancer patients were integratively analyzed to screen Rab proteins and potential immune-related signaling pathways. Imaging and various biochemical assays were performed in Jurkat T cells, splenocytes, and human peripheral blood mononuclear cells (PBMCs). Rab37 knockout mice and specimens of lung cancer patients were used to validate the concept.ResultsHere, we identify novel mechanisms of intracellular trafficking and plasma membrane presentation of PD-1 mediated by Rab37 small GTPase to sustain T cell exhaustion, thereby leading to poor patient outcome. PD-1 colocalized with Rab37-specific vesicles of T cells in a GTP-dependent manner whereby Rab37 mediated dynamic trafficking and membrane presentation of PD-1. However, glycosylation mutant PD-1 delayed cargo recruitment to the Rab37 vesicles, thus stalling membrane presentation. Notably, T cell proliferation and activity were upregulated in tumor-infiltrating T cells from the tumor-bearing Rab37 knockout mice compared to those from wild type. Clinically, the multiplex immunofluorescence-immunohistochemical assay indicated that patients with high Rab37+/PD-1+/TIM3+/CD8+ tumor infiltrating T cell profile correlated with advanced tumor stages and poor overall survival. Moreover, human PBMCs from patients demonstrated high expression of Rab37, which positively correlated with elevated levels of PD-1+ and TIM3+ in CD8+ T cells exhibiting reduced tumoricidal activity.ConclusionsOur results provide the first evidence that Rab37 small GTPase mediates trafficking and membrane presentation of PD-1 to sustain T cell exhaustion, and the tumor promoting function of Rab37/PD-1 axis in T cells of TME in lung cancer. The expression profile of Rab37high/PD-1high/TIM3high in tumor-infiltrating CD8+ T cells is a biomarker for poor prognosis in lung cancer patients.

Requirement of microtubules for secretion of a micronemal protein CpTSP4 in the invasive stage of the apicomplexan Cryptosporidium parvum.

The zoonotic Cryptosporidium parvum is a global contributor to infantile diarrheal diseases and opportunistic infections in immunocompromised or weakened individuals. Like other apicomplexans, it possesses several specialized secretory organelles, including micronemes, rhoptry, and dense granules. However, the understanding of cryptosporidial micronemal composition and secretory pathway remains limited. Here, we report a new micronemal protein in C. parvum, namely, thrombospondin (TSP)-repeat domain-containing protein-4 (CpTSP4), providing insights into these ambiguities. Immunostaining and enzyme-linked assays show that CpTSP4 is prestored in the micronemes of unexcysted sporozoites but secreted during sporozoite excystation, gliding, and invasion. In excysted sporozoites, CpTSP4 is also distributed on the two central microtubules unique to Cryptosporidium. The secretion and microtubular distribution could be completely blocked by the selective kinesin-5 inhibitors SB-743921 and SB-715992, resulting in the accumulation of CpTSP4 in micronemes. These support the kinesin-dependent microtubular trafficking of CpTSP4 for secretion. We also localize γ-tubulin, consistent with kinesin-dependent anterograde trafficking. Additionally, recombinant CpTSP4 displays nanomolar binding affinity to the host cell surface, for which heparin acts as one of the host ligands. A novel heparin-binding motif is identified and validated biochemically for its contribution to the adhesive property of CpTSP4 by peptide competition assays and site-directed mutagenesis. These findings shed light on the mechanisms of intracellular trafficking and secretion of a cryptosporidial micronemal protein and the interaction of a TSP-family protein with host cells.IMPORTANCECryptosporidium parvum is a globally distributed apicomplexan parasite infecting humans and/or animals. Like other apicomplexans, it possesses specialized secretory organelles in the zoites, in which micronemes discharge molecules to facilitate the movement and invasion of zoites. Although past and recent studies have identified several proteins in cryptosporidial micronemes, our understanding of the composition, secretory pathways, and domain-ligand interactions of micronemal proteins remains limited. This study identifies a new micronemal protein, namely, CpTSP4, that is discharged during excystation, gliding, and invasion of C. parvum sporozoites. The CpTSP4 secretion depends on the intracellular trafficking on the two Cryptosporidium-unique microtubes that could be blocked by kinesin-5/Eg5 inhibitors. Additionally, a novel heparin-binding motif is identified and biochemically validated, which contributes to the nanomolar binding affinity of CpTSP4 to host cells. These findings indicate that kinesin-dependent microtubular trafficking is critical to CpTSP4 secretion, and heparin/heparan sulfate is one of the ligands for this micronemal protein.

Beyond nanoparticle-based oral drug delivery: transporter-mediated absorption and disease targeting.

Various strategies at the microscale/nanoscale have been developed to improve oral absorption of therapeutics. Among them, gastrointestinal (GI)-transporter/receptor-mediated nanosized drug delivery systems (NDDSs) have drawn attention due to their many benefits, such as improved water solubility, improved chemical/physical stability, improved oral absorption, and improved targetability of their payloads. Their therapeutic potential in disease animal models (e.g., solid tumors, virus-infected lungs, metastasis, diabetes, and so on) has been investigated, and could be expanded to disease targeting after systemic/lymphatic circulation, although the detailed paths and mechanisms of endocytosis, endosomal escape, intracellular trafficking, and exocytosis through the epithelial cell lining in the GI tract are still unclear. Thus, this review summarizes and discusses potential GI transporters/receptors, their absorption and distribution, in vivo studies, and potential sequential targeting (e.g., oral absorption and disease targeting in organs/tissues).

Attachment, Entry, and Intracellular Trafficking of Classical Swine Fever Virus.

Classical swine fever virus (CSFV), which is a positive-sense, single-stranded RNA virus with an envelope, is a member of the Pestivirus genus in the Flaviviridae family. CSFV causes a severe and highly contagious disease in pigs and is prevalent worldwide, threatening the pig farming industry. The detailed mechanisms of the CSFV life cycle have been reported, but are still limited. Some receptors and attachment factors of CSFV, including heparan sulfate (HS), laminin receptor (LamR), complement regulatory protein (CD46), MER tyrosine kinase (MERTK), disintegrin, and metalloproteinase domain-containing protein 17 (ADAM17), were identified. After attachment, CSFV internalizes via clathrin-mediated endocytosis (CME) and/or caveolae/raft-dependent endocytosis (CavME). After internalization, CSFV moves to early and late endosomes before uncoating. During this period, intracellular trafficking of CSFV relies on components of the endosomal sorting complex required for transport (ESCRT) and Rab proteins in the endosome dynamics, with a dependence on the cytoskeleton network. This review summarizes the data on the mechanisms of CSFV attachment, internalization pathways, and intracellular trafficking, and provides a general view of the early events in the CSFV life cycle.

Abstract 5875: Rab37 mediates intracellular trafficking and membrane presentation of PD-1 in T cells to foster an immunosuppressive microenvironment in lung cancer

Abstract Programmed cell death protein 1 (PD-1) expressed on the surface of CD8+ T cells is known as an immune checkpoint protein. High expression of PD-1 leads to T-cell dysfunction in the tumor microenvironment (TME). Our published studies show that Rab37 small GTPase-mediated vesicular trafficking of cell surface proteins and cytokines is critical in immunosuppressive TME. Here, we identify novel mechanisms of intracellular trafficking and plasma membrane presentation of PD-1 mediated by Rab37. Confocal immunofluorescence (IF) and vesicles isolation data demonstrated that PD-1 colocalized with Rab37-specific vesicles in T cells in a GTP-dependent manner. Total internal reflection fluorescence imaging, membrane fractionation, and flow cytometric analyses confirmed the dynamic trafficking and membrane presentation of PD-1 by Rab37. In addition, sucrose density gradient centrifugation and IF revealed that the level of glycosylated wild-type PD-1 in membrane compartment was more than glycosylation mutant PD-1, suggesting that glycosylation mutant PD-1 delayed in recruitment to the Rab37 vesicles and thus stalled in membrane presentation. Furthermore, T cell proliferation and activity were downregulated in Rab37 wild-type splenocytes co-cultured with cancer cells compared to those from Rab37 knockout mice using various T cell functional assays. Clinically, the multiplex IF-immunohistochemical assay indicated that the tumor infiltrated Rab37+/PD1+/TIM-3+/CD8+ exhaustion T cells were more in late staged lung cancer patients compared to those in early staged patients. Importantly, patients with Rab37+/PD-1+/TIM-3+/CD8+ tumor infiltrated T cells expression profile correlated with poor overall survival. Our results provide first trafficking mode of PD-1 mediated by Rab37 small GTPase and novel evidence of the tumor promoting function of Rab37/PD-1 axis in T cells of the tumor microenvironment. Citation Format: Wan-Ting Kuo, I-Ying Kuo, Shih-Ting Wu, Wu-Chou Su, Yi-Ching Wang. Rab37 mediates intracellular trafficking and membrane presentation of PD-1 in T cells to foster an immunosuppressive microenvironment in lung cancer. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5875.

Abstract 322: Electron microscopy using electron dense genetic tag elucidates novel extracellular vesicle biogenesis in non-small cell lung cancer

Abstract Extracellular vesicles (EVs) act as key mediators of cell-to-cell communication in several pathophysiological processes including cancer. EVs inherit a cocktail of molecular cues (mRNAs, miRNAs, ncRNAs, proteins, metabolites, etc.) from cancer cells that mediate tumor microenvironment changes to facilitate cancer progression and metastasis. The heterogenous EV populations and their content dynamically change during cancer, which is why, there has been great progress in utilizing them as prognostic and diagnostic biomarkers. However, harnessing EVs for therapeutic applications requires greater understanding of the fundamental EV biology such as their origin, intracellular trafficking, cargo loading, and export mechanisms. Here, we utilized a genetically-encoded electron dense tag, APEX2 (an engineered peroxidase derived from soybean ascorbate peroxidase) and electron microscopy to elucidate that EVs, in the form of multivesicular bodies, can also originate at the trans-Golgi interface. We also identified a novel subpopulation of EVs, which expressed a classic trans-Golgi network protein (referred as TGNP) on their surface. While EVs of late endosomal origin are well known, and there are reports of mitochondrial origin as well, a trans-Golgi EV biogenesis pathway has not been reported before. Intriguingly, we determined that certain cells hijack the trans-Golgi pathway for EV biogenesis, which we hypothesize contributes to differential cancer cell EV cargo and phenotypic consequences. Initially, using confocal microscopy in non-small cell lung cancer (NSCLC) cells, we identified that some of the tetraspanins historically thought to be involved in late endosomal trafficking were instead enriched in the trans-Golgi compartments. We found that this colocalization pattern was conserved in several other NSCLC cell lines. To further elucidate this phenomenon at the trans-Golgi interface, we tagged TGNP to APEX2. After verifying the expression and localization of TGNP-APEX2 by immunostaining, we performed electron microscopy. TGNP-APEX2 was found to be present on the membrane of intraluminal vesicles inside multivesicular bodies. Interestingly, the multivesicular bodies containing TGNP-APEX2 vesicles were not of late endosome origin. Furthermore, TGNP was also found to be present in EVs of all NSCLC cell lines tested. Inhibiting Golgi trafficking using small molecule inhibitors (Brefeldin A) significantly reduced EV secretion and levels of trans-Golgi-enriched tetraspanins in the EV lysates. The data elucidates that EVs can also originate from trans-Golgi interface and identified novel subpopulation that expresses TGNP. The broader questions regarding the functional relevance of trans-Golgi-mediated EV biogenesis, the type of cargo exported, and mediators of loading and export in the context of cancer are currently under investigation. Citation Format: Ikjot Singh Sohal, Sydney N. Shaw, Andrea L. Kasinski. Electron microscopy using electron dense genetic tag elucidates novel extracellular vesicle biogenesis in non-small cell lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 322.

Fatty Acid-Binding Protein 5 Gene Deletion Enhances Nicotine-Conditioned Place Preference: Illuminating the Putative Gateway Mechanisms.

Emerging evidence indicates that the endogenous cannabinoid system modulates the behavioral and physiological effects of nicotine. Fatty acid-binding proteins (FABPs) are among the primary intracellular trafficking mechanisms of endogenous cannabinoids, such as anandamide. To this end, changes in FABP expression may similarly impact the behavioral manifestations associated with nicotine, particularly its addictive properties. FABP5 +/+ and FABP5 -/- mice were tested for nicotine-conditioned place preference (CPP) at two different doses (0.1 or 0.5 mg/kg). The nicotine-paired chamber was assigned as their least preferred chamber during preconditioning. Following 8 days of conditioning, the mice were injected with either nicotine or saline. The mice were allowed to access to all the chambers on the test day, and their times spent in the drug chamber on the preconditioning versus the test days were used to examine the drug preference score. The CPP results showed that the FABP5 -/- mice displayed a higher place preference for 0.1 mg/kg nicotine than the FABP5 +/+ mice, while no CPP difference was observed for 0.5 mg/kg nicotine between the genotypes. In conclusion, FABP5 plays an important role in regulating nicotine place preference. Further research is warranted to identify the precise mechanisms. The results suggest that dysregulated cannabinoid signaling may impact nicotine-seeking behavior.

Converging links between adult-onset neurodegenerative Alzheimer's disease and early life neurodegenerative neuronal ceroid lipofuscinosis?

Evidence from genetics and from analyzing cellular and animal models have converged to suggest links between neurodegenerative disorders of early and late life. Here, we summarize emerging links between the most common late life neurodegenerative disease, Alzheimer's disease, and the most common early life neurodegenerative diseases, neuronal ceroid lipofuscinoses. Genetic studies reported an overlap of clinically diagnosed Alzheimer's disease and mutations in genes known to cause neuronal ceroid lipofuscinoses. Accumulating data strongly suggest dysfunction of intracellular trafficking mechanisms and the autophagy-endolysosome system in both types of neurodegenerative disorders. This suggests shared cytopathological processes underlying these different types of neurodegenerative diseases. A better understanding of the common mechanisms underlying the different diseases is important as this might lead to the identification of novel targets for therapeutic concepts, the transfer of therapeutic strategies from one disease to the other and therapeutic approaches tailored to patients with specific mutations. Here, we review dysfunctions of the endolysosomal autophagy pathway in Alzheimer's disease and neuronal ceroid lipofuscinoses and summarize emerging etiologic and genetic overlaps.

Epidermal stratification requires retromer-mediated desmoglein-1 recycling.

Sorting transmembrane cargo is essential for tissue development and homeostasis. However, mechanisms of intracellular trafficking in stratified epidermis are poorly understood. Here, we identify an interaction between the retromer endosomal trafficking component, VPS35, and the desmosomal cadherin, desmoglein-1 (Dsg1). Dsg1 is specifically expressed in stratified epidermis and, when properly localized on the plasma membrane of basal keratinocytes, promotes stratification. We show that the retromer drives Dsg1 recycling from the endo-lysosomal system to the plasma membrane to support human keratinocyte stratification. The retromer-enhancing chaperone, R55, promotes the membrane localization of Dsg1 and a trafficking-deficient mutant associated with a severe inflammatory skin disorder, enhancing its ability to promote stratification. In the absence of Dsg1, retromer association with and expression of the glucose transporter GLUT1 increases, exposing a potential link between Dsg1 deficiency and epidermal metabolism. Our work provides evidence for retromer function in epidermal regeneration, identifying it as a potential therapeutic target.

Time-resolved proximity labeling of protein networks associated with ligand-activated EGFR.

SUMMARYLigand binding to the EGF receptor (EGFR) triggers multiple signal-transduction processes and promotes endocytosis of the receptor. The mechanisms of EGFR endocytosis and its cross-talk with signaling are poorly understood. Here, we combine peroxidase-catalyzed proximity labeling, isobaric peptide tagging, and quantitative mass spectrometry to define the dynamics of the proximity proteome of ligand-activated EGFR. Using this approach, we identify a network of signaling proteins, which remain associated with the receptor during its internalization and trafficking through the endosomal system. We show that Trk-fused gene (TFG), a protein known to function at the endoplasmic reticulum exit sites, is enriched in the proximity proteome of EGFR in early/sorting endosomes and localized in these endosomes and demonstrate that TFG regulates endosomal sorting of EGFR. This study provides a comprehensive resource of time-dependent nanoscale environment of EGFR, thus opening avenues to discovering new regulatory mechanisms of signaling and intracellular trafficking of receptor tyrosine kinases.

Perturbing the Normal Level of SIDT1 Suppresses the Naked ASO Effect.

Although antisense oligonucleotide (ASO) therapeutics can be taken up by living cells without carrier molecules, a large part of incorporated ASOs are trapped in the endosomes and do not exert therapeutic effects. To improve their therapeutic effects, it would be important to elucidate the mechanism of cellular uptake and intracellular trafficking of ASOs. In this study, we investigated how SIDT1 affects cellular uptake and intracellular trafficking of ASOs. Fluorescence microscopic analysis suggested that most of naked ASOs are trafficked to the lysosomes via the endosomes. The data obtained from flow cytometry and fluorescence microscopy together showed that although the SIDT1 level barely affects the total cellular uptake of ASOs, it appears to affect the intracellular trafficking of ASOs. We also showed that SIDT1 exists mainly in the endoplasmic reticulum and that perturbing the normal level of SIDT1 suppresses the antisense effect of the naked ASO targeting miR-16.

FYVE2, a phosphatidylinositol 3-phosphate effector, interacts with the COPII machinery to control autophagosome formation in Arabidopsis.

Autophagy is an intracellular trafficking mechanism by which cytosolic macromolecules and organelles are sequestered into autophagosomes for degradation inside the vacuole. In various eukaryotes including yeast, metazoans, and plants, the precursor of the autophagosome, termed the phagophore, nucleates in the vicinity of the endoplasmic reticulum (ER) with the participation of phosphatidylinositol 3-phosphate (PI3P) and the coat protein complex II (COPII). Here we show that Arabidopsis thaliana FYVE2, a plant-specific PI3P-binding protein, provides a functional link between the COPII machinery and autophagy. FYVE2 interacts with the small GTPase Secretion-associated Ras-related GTPase 1 (SAR1), which is essential for the budding of COPII vesicles. FYVE2 also interacts with ATG18A, another PI3P effector on the phagophore membrane. Fluorescently tagged FYVE2 localized to autophagic membranes near the ER and was delivered to vacuoles. SAR1 fusion proteins were also targeted to the vacuole via FYVE2-dependent autophagy. Either mutations in FYVE2 or the expression of dominant-negative mutant SAR1B proteins resulted in reduced autophagic flux and the accumulation of autophagic organelles. We propose that FYVE2 regulates autophagosome biogenesis through its interaction with ATG18A and the COPII machinery, acting downstream of ATG2.

Polycation-Mediated Transfection: Mechanisms of Internalization and Intracellular Trafficking.

Polyplex-mediated gene transfection is now in its' fourth decade of serious research, but the promise of polyplex-mediated gene therapy has yet to fully materialize. Only approximately one in a million applied plasmids actually expresses. A large part of this is due to an incomplete understanding of the mechanism of polyplex transfection. There is an assumption that internalization must follow a canonical mechanism of receptor mediated endocytosis. Herein, we present arguments that untargeted (and most targeted) polyplexes do not utilize these routes. By incorporating knowledge of syndecan-polyplex interactions, we can show that syndecans are the "target" for polyplexes. Further, it is known that free polycations (which disrupt cell-membranes by acid-catalyzed hydrolysis of phospholipid esters) are necessary for (untargeted) endocytosis. This can be incorporated into the model to produce a novel mechanism of endocytosis, which fits the observed phenomenology. After membrane translocation, polyplex containing vesicles reach the endosome after diffusing through the actin mesh below the cell membrane. From there, they are acidified and trafficked toward the lysosome. Some polyplexes are capable of escaping the endosome and unpacking, while others are not. Herein, it is argued that for some polycations, as acidification proceeds the polyplexes excluding free polycations, which disrupt the endosomal membrane by acid-catalyzed hydrolysis, allowing the polyplex to escape. The polyplex's internal charge ratio is now insufficient for stability and it releases plasmids which diffuse to the nucleus. A small proportion of these plasmids diffuse through the nuclear pore complex (NPC), with aggregation being the major cause of loss. Those plasmids that have diffused through the NPC will also aggregate, and this appears to be the reason such a small proportion of nuclear plasmids express mRNA. Thus, the structural features which promote unpacking in the endosome and allow for endosomal escape can be determined, and better polycations can be designed.

Methods for Functional Characterization of FcRn Interactions with Therapeutic Antibodies and Fc-Fusion Proteins.

The neonatal Fc receptor (FcRn) plays a key role in determining the pharmacokinetic behavior of therapeutic monoclonal antibodies (mAbs). FcRn-mediated intracellular trafficking mechanisms extend the half-lives of mAbs by rescuing them from lysosomal degradation and contribute to their transportation from the vascular space to tissue compartments such as placenta and mucosal surfaces. It is important to characterize the FcRn interactions of therapeutic mAbs and Fc-fusion proteins due to its potential impact on their in vivo pharmacokinetic properties such as clearance and half-life. In this chapter, we describe protocols for two cell-based assays that measure the total function of FcRn which involves pH-dependent association and dissociation with IgG-Fc, as well as FcRn-mediated intracellular trafficking parameters. These assays are suitable for characterization of FcRn interactions with therapeutic mAbs and Fc-fusion proteins for the purpose of assessing lot-to-lot consistency and the structural and functional integrity of the Fc domain. In addition, they may serve as cost-effective screening tools for the evaluation of mAb-based drug candidates during lead selection and optimization for desired pharmacokinetic properties.

Exploring the transformability of polymer-lipid hybrid nanoparticles and nanomaterial-biology interplay to facilitate tumor penetration, cellular uptake and intracellular targeting of anticancer drugs

ABSTRACT Background Successful delivery of anticancer drugs to intracellular targets requires different properties of the nanocarrier to overcome multiple transport barriers. However, few nanocarrier systems, to date, possess such properties, despite knowledge about the biological fate of inorganic and polymeric nanocarriers in relation to their fixed size, shape and surface properties. Herein, a polymer-lipid hybrid nanoparticle (PLN) system is described with size and shape transformability and its mechanisms of cellular uptake and intracellular trafficking are studied. Methods Pharmaceutical lipids were screened for use in transformable PLN. Mechanisms of cellular uptake and the role of fatty acid-binding proteins in intracellular trafficking of PLN were investigated in breast cancer cells. Intra-tumoral penetration and retention of doxorubicin (DOX) were evaluated by confocal microscopy. Results The lead PLNs showed time-dependent size reduction and shape change from spherical to spiky shape. This transformability of PLNs and lipid trafficking pathways facilitated intracellular transport of DOX-loaded PLN (DOX-PLN) into mitochondria and nuclei. DOX-PLN significantly increased DOX penetration and retention over free DOX or non-transformable liposomal DOX particles at 4 h post-intravenous administration. Conclusion Transformability of PLN and lipid-biology interplay can be exploited to design new nanocarriers for effective drug delivery to tumor cells and intracellular targets.

Rickettsia-host interaction: strategies of intracytosolic host colonization

ABSTRACTBacterial infection is a highly complex biological process involving a dynamic interaction between the invading microorganism and the host. Specifically, intracellular pathogens seize control over the host cellular processes including membrane dynamics, actin cytoskeleton, phosphoinositide metabolism, intracellular trafficking and immune defense mechanisms to promote their host colonization. To accomplish such challenging tasks, virulent bacteria deploy unique species-specific secreted effectors to evade and/or subvert cellular defense surveillance mechanisms to establish a replication niche. However, despite superficially similar infection strategies, diverse Rickettsia species utilize different effector repertoires to promote host colonization. This review will discuss our current understandings on how different Rickettsia species deploy their effector arsenal to manipulate host cellular processes to promote their intracytosolic life within the mammalian host.

High-Content Analysis Monitoring Intracellular Trafficking and Replication of Mycobacterium tuberculosis Inside Host Cells.

Mycobacterium tuberculosis is able to colonize, persist, and massively replicate in host cells, such as phagocytes and epithelial cells. The intracellular stage of the bacteria is critical to the development of tuberculosis pathogenesis. The detailed mechanisms of intracellular trafficking of the bacillus are not fully understood and require further investigations. Therefore, increasing the knowledge of this process will help to develop therapeutic tools that will lower the burden of tuberculosis. M. tuberculosis is genetically tractable and tolerates the expression of heterologous fluorescent proteins. Thus, the intracellular distribution of the bacteria expressing fluorescent tracers can be easily defined using confocal microscopy. Advances in imaging techniques and images-based analysis allow the rapid quantification of biological objects in complex environments. In this chapter, we detailed high-content / high-throughput imaging methods to track the bacillus within host cell settings.

Separation-related rapid nuclear transport of DNA/RNA heteroduplex oligonucleotide: unveiling distinctive intracellular trafficking

DNA/RNA heteroduplex oligonucleotide (HDO), composed of DNA/locked nucleic acid (LNA) antisense oligonucleotide (ASO) and complementary RNA, is a next-generation antisense therapeutic agent. HDO is superior to the parental ASO in delivering to target tissues, and it exerts a more potent gene-silencing effect. In this study, we aimed to elucidate the intracellular trafficking mechanism of HDO-dependent gene silencing. HDO was more preferably transferred to the nucleus after transfection compared to the parental ASO. To determine when and where HDO is separated into the antisense strand (AS) and complementary strand (CS), we performed live-cell time-lapse imaging and fluorescence resonance energy transfer (FRET) assays. These assays demonstrated that HDO had a different intracellular trafficking mechanism than ASO. After endocytosis, HDO was separated in the early endosomes, and both AS and CS were released into the cytosol. AS was more efficiently transported to the nucleus than CS. Separation, endosomal release, and initiation of nuclear transport were a series of time-locked events occurring at a median of 30 s. CS cleavage was associated with efficient nuclear distribution and gene silencing in the nucleus. Understanding the unique intracellular silencing mechanisms of HDO will help us design more efficient drugs and might also provide insight into innate DNA/RNA cellular biology.

In Silico Docking of Vitamin E Isomers on Transport Proteins.

Vitamin E is comprised of α, β, γ and δ-tocopherols (Ts) and α, β, γ and δ- tocotrienols (T3s). Vitamin E has neuroprotective antioxidant, anti-cancer, and cholesterol-lowering effects. Intracellular trafficking of these isomers remains largely unknown, except for αT which is selectively transported by αT transfer protein (αTTP). This study aimed to determine the binding of vitamin E isomers on transport proteins using in silico docking. Transport proteins were selected using AmiGo Gene Ontology tool based on the same molecular function annotation as αTTP. Protein structures were obtained from the Protein Data Bank. Ligands structures were obtained from ZINC database. In silico docking was performed using SwissDock. A total of 6 transport proteins were found: SEC14-like protein 2, glycolipid transfer protein (GLTP), pleckstrin homology domain-containing family A member 8, collagen type IV alpha-3-binding protein, ceramide-1-phosphate transfer protein and afamin. Compared with other transport proteins, αTTP had the highest affinities for all isomers except βT3. Binding order of vitamin E isomers toward αTTP was γT > βT > αT > δT > αT3 > γT3 > δT3 > βT3. GLTP had a higher affinity for tocotrienols than tocopherols. βT3 bound stronger to GLTP than αTTP. αTTP remained as the most preferred transport protein for most of the isomers. The binding affinity of αT toward αTTP was not the highest than other isomers suggested that other intracellular trafficking mechanisms of these isomers may exist. GLTP may mediate the intracellular transport of tocotrienols, especially βT3. Improving the bioavailability of these isomers may enhance their beneficial effects to human.