Intracellular Transport Mechanisms Research Articles

Pharmacokinetics and antioxidant activity of dihydrocaffeic acid grafted chitosan nanomicelles loaded with chicoric acid in broilers

Chicoric acid (CA) is a natural nutrient found in plants, showcasing diverse biological activities, including anti-inflammatory and antioxidant properties. Despite its valuable properties, CA faces limitations in bioavailability and susceptibility to oxidative breakdown during utilization. Previous research introduced synthesized dihydrocaffeic acid grafted chitosan self-assembled nanomicelles (DA-g-CS), demonstrating its potential to enhance CA absorption. This study aims to investigate the pharmacokinetics, tissue distribution, and antioxidant activity of both CA and DA-g-CS loaded CA (DA-g-CS/CA) in broilers. An IPEC-J2 cell model was established and evaluated to delve deeper into the transport mechanism and antioxidant potential. The in vivo pharmacokinetic analysis in broilers highlighted a substantial difference: the maximum plasma concentration (Cmax) of DA-g-CS/CA exceeded CA by 2.6-fold, yielding a notable increased relative bioavailability to 214%. This evidence underscores the significant enhancement in CA's oral absorption, facilitated by DA-g-CS. The collective evaluation outcomes affirm the successful development of the cell model, indicating its suitability for drug transporter experiments. The findings from the intestinal transit analysis revealed that both CA and DA-g-CS/CA underwent passive entry into IPEC-J2 cells. Notably, the cellular uptake rate of DA-g-CS loaded with CA was significantly amplified, reaching 2.1 times higher than that of CA alone. Intracellular transport mechanisms involved microtubules, lysosomes, and the endoplasmic reticulum, with an additional pathway involving the endoplasmic reticulum observed specifically for DA-g-CS/CA, distinguishing it from CA. Moreover, the results from both in vivo and in vitro antioxidant assessments highlight the potent antioxidant activity of DA-g-CS/CA, showcasing its efficacy in preventing and treating cellular damage induced by oxidative stress. In summary, these findings underscore the significant enhancement of CA's efficacy facilitated by DA-g-CS, establishing a robust theoretical foundation for the prospective application of CA within livestock and poultry farming.

Cytokinin: From autoclaved DNA to two-component signaling.

Since its first identification in the 1950s as a regulator of cell division, cytokinin has been linked to many physiological processes in plants, spanning growth and development and various responses to the environment. Studies from the last two and one-half decades have revealed the pathways underlying the biosynthesis and metabolism of cytokinin and have elucidated the mechanisms of its perception and signaling, which reflects an ancient signaling system evolved from two-component elements in bacteria. Mutants in the genes encoding elements involved in these processes have helped refine our understanding of cytokinin functions in plants. Further, recent advances have provided insight into the mechanisms of intracellular and long-distance cytokinin transport and the identification of several proteins that operate downstream of cytokinin signaling. Here, we review these processes through a historical lens, providing an overview of cytokinin metabolism, transport, signaling, and functions in higher plants.

Nicotianamine: A Key Player in Metal Homeostasis and Hyperaccumulation in Plants

Nicotianamine (NA) is a low-molecular-weight N-containing metal-binding ligand, whose accumulation in plant organs changes under metal deficiency or excess. Although NA biosynthesis can be induced in vivo by various metals, this non-proteinogenic amino acid is mainly involved in the detoxification and transport of iron, zinc, nickel, copper and manganese. This review summarizes the current knowledge on NA biosynthesis and its regulation, considers the mechanisms of NA secretion by plant roots, as well as the mechanisms of intracellular transport of NA and its complexes with metals, and its role in radial and long-distance metal transport. Its role in metal tolerance is also discussed. The NA contents in excluders, storing metals primarily in roots, and in hyperaccumulators, accumulating metals mainly in shoots, are compared. The available data suggest that NA plays an important role in maintaining metal homeostasis and hyperaccumulation mechanisms. The study of metal-binding compounds is of interdisciplinary significance, not only regarding their effects on metal toxicity in plants, but also in connection with the development of biofortification approaches to increase the metal contents, primarily of iron and zinc, in agricultural plants, since the deficiency of these elements in food crops seriously affects human health.

Non-Vesicular Lipid Transport Machinery in Leishmania donovani: Functional Implications in Host-Parasite Interaction.

Eukaryotic cells have distinct membrane-enclosed organelles, each with a unique biochemical signature and specialized function. The unique identity of each organelle is greatly governed by the asymmetric distribution and regulated intracellular movement of two important biomolecules, lipids, and proteins. Non-vesicular lipid transport mediated by lipid-transfer proteins (LTPs) plays essential roles in intra-cellular lipid trafficking and cellular lipid homeostasis, while vesicular transport regulates protein trafficking. A comparative analysis of non-vesicular lipid transport machinery in protists could enhance our understanding of parasitism and basis of eukaryotic evolution. Leishmania donovani, the trypanosomatid parasite, greatly depends on receptor-ligand mediated signalling pathways for cellular differentiation, nutrient uptake, secretion of virulence factors, and pathogenesis. Lipids, despite being important signalling molecules, have intracellular transport mechanisms that are largely unexplored in L. donovani. We have identified a repertoire of sixteen (16) potential lipid transfer protein (LTP) homologs based on a domain-based search on TriTrypDB coupled with bioinformatics analyses, which signifies the presence of well-organized lipid transport machinery in this parasite. We emphasized here their evolutionary uniqueness and conservation and discussed their potential implications for parasite biology with regards to future therapeutic targets against visceral leishmaniasis.

Spatial and temporal dynamics of ATP synthase from mitochondria toward the cell surface

Ectopic ATP synthase complex (eATP synthase), located on cancer cell surface, has been reported to possess catalytic activity that facilitates the generation of ATP in the extracellular environment to establish a suitable microenvironment and to be a potential target for cancer therapy. However, the mechanism of intracellular ATP synthase complex transport remains unclear. Using a combination of spatial proteomics, interaction proteomics, and transcriptomics analyses, we find ATP synthase complex is first assembled in the mitochondria and subsequently delivered to the cell surface along the microtubule via the interplay of dynamin-related protein 1 (DRP1) and kinesin family member 5B (KIF5B). We further demonstrate that the mitochondrial membrane fuses to the plasma membrane in turn to anchor ATP syntheses on the cell surface using super-resolution imaging and real-time fusion assay in live cells. Our results provide a blueprint of eATP synthase trafficking and contribute to the understanding of the dynamics of tumor progression.

Non-vesicular Lipid Transport Machinery in Trichomonas vaginalis: Novel Drug Targets against Trichomoniasis.

Eukaryotic cells have separate membrane-enclosed organelles with distinct biochemical identities and specialized functions. The unique characteristics of each organelle are regulated by asymmetric distribution and intra-cellular trafficking of two important biomolecules, proteins and lipids. Non-vesicular lipid transport facilitated by lipid transfer proteins performs essential roles in intracellular lipid trafficking and homeostasis, while vesicular transport regulates protein trafficking. Comparative analysis of lipid transport machinery in protists could help us to understand the basis of parasitism and insight into eukaryotic evolution. Trichomonas vaginalis, a parasitic protist, greatly depends on receptor-ligand-mediated signaling pathways for cellular differentiation, nutrient uptake, secretion of virulence factors, and pathogenesis. Lipids, despite being key molecules of signaling cascades, have mechanisms of intracellular transport that are largely unexplored in T. vaginalis. We have identified a repertoire of seventeen potential lipid transfer protein (LTP) homologs in T. vaginalis based on a domain-based search on TrichDB (genome database of Trichomonas) coupled with bioinformatics analyses, which indicates the presence of well-organized lipid transport machinery in this parasite. We emphasized here their evolutionary uniqueness and conservation and discussed their potential implications for parasite biology in regard to future therapeutic targets against Trichomoniasis.

The intracellular fate and transport mechanism of shape, size and rigidity varied nanocarriers for understanding their oral delivery efficiency

Nanocarriers have become an effective strategy to overcome epithelial absorption barriers. During the absorption process, the endocytosis mechanisms, cell internalization pathways, and transport efficiency of nanocarriers are greatly impacted by their physical properties. To understand the relationship between physical properties of nanocarriers and their abilities overcoming multiple absorption barriers, nanocarriers with variable physical properties were prepared via self-assembly of hydrolyzed α-lactalbumin peptide fragments. The impacts of size, shape, and rigidity of nanocarriers on epithelial cells endocytosis mechanisms, internalization pathways, transport efficiency, and bioavailability were studied systematically. The results showed that nanospheres were mainly internalized via clathrin-mediated endocytosis, which was then locked in lysosomes and degraded enzymatically in cytoplasm. While macropinocytosis was the primary pathway of nanotubes and transported to the endoplasmic reticulum and Golgi apparatus, resulting in a high drug concentration and sustained release in cytoplasm. Besides, nanotubes can overcome the multi-drug resistance by inhibiting the P-glycoprotein efflux. Furthermore, nanotubes can open intercellular tight-junctions instantaneously and reversibly, which promotes transport into blood circulation. The aqueous solubility of hydrophobic bioactive mangiferin (Mgf) was improved by nanocarriers. Most importantly, the bioavailability of Mgf was the highest for cross-linked short nanotube (CSNT) which outperformed free Mgf and other formulations by in vivo pharmacokinetic studies. Finally, Mgf-loaded CSNT showed an excellent therapeutic efficiency in vivo for the intervention of streptozotocin-induced diabetes. These results indicate that cross-linked α-lactalbumin nanotubes could be an effective nanocarrier delivery system for improving the epithelium cellular absorption and bioavailability of hydrophobic bioactive compounds.

Nanocarriers for the delivery of antibiotics into cells against intracellular bacterial infection.

The barrier function of host cells enables intracellular bacteria to evade the lethality of the host immune system and antibiotics, thereby causing chronic and recurrent infections that seriously threaten human health. Currently, the main clinical strategy for the treatment of intracellular bacterial infections involves the use of long-term and high-dose antibiotics. However, insufficient intracellular delivery of antibiotics along with various resistance mechanisms not only weakens the efficacy of current therapies but also causes serious adverse drug reactions, further increasing the disease and economic burden. Improving the delivery efficiency, intracellular accumulation, and action time of antibiotics remains the most economical and effective way to treat intracellular bacterial infections. The rapid development of nanotechnology provides a strategy to efficiently deliver antibiotics against intracellular bacterial infections into cells. In this review, we summarize the types of common intracellular pathogens, the difficulties faced by antibiotics in the treatment of intracellular bacterial infections, and the research progress of several types of representative nanocarriers for the delivery of antibiotics against intracellular bacterial infections that have emerged in recent years. This review is expected to provide a reference for further elucidating the intracellular transport mechanism of nanocarrier-drug complexes, designing safer and more effective nanocarriers and establishing new strategies against intracellular bacterial infection.

In Vitro Methods to Study the Golgi Apparatus Role in Proteoglycan and Glycosaminoglycan Synthesis.

Subcellular fractionation is an introductory step in a variety of experimental approaches designed to study intracellular components, like membranes and organelle systems. Subcellular fractions enriched in membranes of the Golgi apparatus of mammalian cells have been isolated to address localization and activity of proteins, including enzymes, to study intracellular membrane transport mechanisms, and to reconstitute in vitro cellular processes associated with the Golgi apparatus. Here, I describe methods to purify Golgi membranes by subcellular fractionation, to assay nucleotide sulfate (PAPS) uptake into Golgi vesicles, and to measure sulfate incorporation into in vitro synthesized glycosaminoglycans.

Nano-positioning and tubulin conformation contribute to axonal transport regulation of mitochondria along microtubules

Correct spatiotemporal distribution of organelles and vesicles is crucial for healthy cell functioning and is regulated by intracellular transport mechanisms. Controlled transport of bulky mitochondria is especially important in polarized cells such as neurons that rely on these organelles to locally produce energy and buffer calcium. Mitochondrial transport requires and depends on microtubules that fill much of the available axonal space. How mitochondrial transport is affected by their position within the microtubule bundles is not known. Here, we found that anterograde transport, driven by kinesin motors, is susceptible to the molecular conformation of tubulin in neurons both invitro and invivo. Anterograde velocities negatively correlate with the density of elongated tubulin dimers like guanosine triphosphate (GTP)-tubulin. The impact of the tubulin conformation depends primarily on where a mitochondrion is positioned, either within or at the rim of microtubule bundle. Increasing elongated tubulin levels lowers the number of motile anterograde mitochondria within the microtubule bundle and increases anterograde transport speed at the microtubule bundle rim. We demonstrate that the increased kinesin velocity and density on microtubules consisting of elongated dimers add to the increased mitochondrial dynamics. Our work indicates that the molecular conformation of tubulin contributes to the regulation of mitochondrial motility and as such to the local distribution of mitochondria along axons.

Exploring the cellular uptake of hectorite clay mineral and its drug carrier capabilities

In the last years, the use of clay minerals for pharmaceutical purposes has increased due to their interesting properties. Hectorite (Ht) is a clay belonging to the smectite group which has attracted attention for applications in biology, tissue engineering and as drug carrier and delivery system. However, the mechanisms involved in Ht cellular uptake and transport into cells, are still unclear. Herein, we used a labeled Ht (Ht/1Cl) to study both the cellular uptake, by confocal laser scanning microscopy, and internalization pathways involved in the cellular uptake, by various endocytosis-inhibiting studies and fluorescence microscopy. These studies highlighted that Ht can penetrate the cellular membrane, localizing mainly in the cytoplasm. The main intracellular transport mechanisms are the ATP-dependent ones and those where filaments and microtubules are involved. Finally, as proof of concept for the potential of Ht as carrier system, we envisaged the covalent grafting of the anticancer molecule methotrexate (MTX), chosen as model, to obtain the Ht-MTX nanomaterial. The covalent linkage was confirmed by several techniques and the morphology of the obtained nanomaterial was imaged by SEM and TEM investigations. The kinetic release of the drug from the Ht-MTX nanomaterial in physiological conditions was studied as well. Furthermore, cytotoxic studies on different cell lines, namely, HL-60, HL-60R, MCF-7, 5637, UMUC3 and RT112 showed that Ht could be a promising material for anticancer therapy.

Induction of Hypoxic Response in Caco-2 Cells Promote the Expression of Genes Involved in SARS-CoV-2 Endocytosis and Transcytosis

In the present manuscript we analyzed the influence of hypoxic response in Caco-2 cells on the expression of genes and miRNAs involved in the mechanisms of intracellular transport of SARS-CoV-2 viral particles, especially endocytosis and transcytosis. With the use of RNA sequencing of Caco-2 cells treated with hypoxia-inducing oxyquinoline derivative, we showed two-fold increase in the expression of the main SARS-CoV-2 receptor ACE2. Expression of the non-canonical receptor TFRC was also elevated. We also observed a significant increase in the expression levels of genes from the low-density lipoprotein (LDL) receptor family, which play a crucial role in the transcytosis: LDLR, LRP1, LRP4, and LRP5. Upregulation of LDLR was coupled with the downregulation of hsa-miR-148a-3p, which can directly bind to LDLR mRNA. Thus, the hypoxic response in Caco-2 cells includes upregulation of genes involved in the mechanisms of endocytosis and transcytosis of SARS-CoV-2 viral particles.

Hitchhiking Across Kingdoms: Cotransport of Cargos in Fungal, Animal, and Plant Cells.

Eukaryotic cells across the tree of life organize their subcellular components via intracellular transport mechanisms. In canonical transport, myosin, kinesin, and dynein motor proteins interact with cargos via adaptor proteins and move along filamentous actin or microtubule tracks. In contrast to this canonical mode, hitchhiking is a newly discovered mode of intracellular transport in which a cargo attaches itself to an already-motile cargo rather than directly associating with a motor protein itself. Many cargos including messenger RNAs, protein complexes, and organelles hitchhike on membrane-bound cargos. Hitchhiking-like behaviors have been shown to impact cellular processes including local protein translation, long-distance signaling, and organelle network reorganization. Here, we review instances of cargo hitchhiking in fungal, animal, and plant cells and discuss the potential cellular and evolutionary importance of hitchhiking in these different contexts.

Chemophoresis engine: A general mechanism of ATPase-driven cargo transport.

Cell polarity regulates the orientation of the cytoskeleton members that directs intracellular transport for cargo-like organelles, using chemical gradients sustained by ATP or GTP hydrolysis. However, how cargo transports are directly mediated by chemical gradients remains unknown. We previously proposed a physical mechanism that enables directed movement of cargos, referred to as chemophoresis. According to the mechanism, a cargo with reaction sites is subjected to a chemophoresis force in the direction of the increased concentration. Based on this, we introduce an extended model, the chemophoresis engine, as a general mechanism of cargo motion, which transforms chemical free energy into directed motion through the catalytic ATP hydrolysis. We applied the engine to plasmid motion in a ParABS system to demonstrate the self-organization system for directed plasmid movement and pattern dynamics of ParA-ATP concentration, thereby explaining plasmid equi-positioning and pole-to-pole oscillation observed in bacterial cells and in vitro experiments. We mathematically show the existence and stability of the plasmid-surfing pattern, which allows the cargo-directed motion through the symmetry-breaking transition of the ParA-ATP spatiotemporal pattern. We also quantitatively demonstrate that the chemophoresis engine can work even under in vivo conditions. Finally, we discuss the chemophoresis engine as one of the general mechanisms of hydrolysis-driven intracellular transport.

Imaging Axonal Transport in Ex Vivo Central and Peripheral Nerves.

Neurones are highly polarized cells with extensive axonal projections that rely on transport of proteins, RNAs, and organelles in a bidirectional manner to remain healthy. This process, known as axonal transport, can be imaged in real time through epifluorescent imaging of fluorescently labeled proteins, organelles, and other cargoes. While this is most conveniently done in primary neuronal cultures, it is more physiologically relevant when carried out in the context of a developed nerve containing both axons and glia. Here we outline how to image axonal transport ex vivo in sciatic and optic nerves, and the fimbria of the fornix. These methods could be altered to image other fluorescently labeled molecules, as well as different mechanisms of intracellular transport.

Tegument Assembly, Secondary Envelopment and Exocytosis.

Alphaherpesvirus tegument assembly, secondary envelopment, and exocytosis processes are understood in broad strokes, but many of the individual steps in this pathway, and their molecular and cell biological details, remain unclear. Viral tegument and membrane proteins form an extensive and robust protein interaction network, such that essentially any structural protein can be deleted, yet particles are still assembled, enveloped, and released from infected cells. We conceptually divide the tegument proteins into three groups: conserved inner and outer teguments that participate in nucleocapsid and membrane contacts, respectively; and 'middle' tegument proteins, consisting of some of the most abundant tegument proteins that serve as central hubs in the protein interaction network, yet which are unique to the alphaherpesviruses. We then discuss secondary envelopment, reviewing the tegument-membrane contacts and cellular factors that drive this process. We place this viral process in the context of cell biological processes, including the endocytic pathway, ESCRT machinery, autophagy, secretory pathway, intracellular transport, and exocytosis mechanisms. Finally, we speculate about potential relationships between cellular defenses against oligomerizing or aggregating membrane proteins and the envelopment and egress of viruses.

Direct imaging of intraflagellar-transport turnarounds reveals that motors detach, diffuse, and reattach to opposite-direction trains

Intraflagellar transport (IFT), a bidirectional intracellular transport mechanism in cilia, relies on the cooperation of kinesin-2 and IFT-dynein motors. In Caenorhabditis elegans chemosensory cilia, motors undergo rapid turnarounds to effectively work together in driving IFT. Here, we push the envelope of fluorescence imaging to obtain insight into the underlying mechanism of motor turnarounds. We developed an alternating dual-color imaging system that allows simultaneous single-molecule imaging of kinesin-II turnarounds and ensemble imaging of IFT trains. This approach allowed direct visualization of motor detachment and reattachment during turnarounds and accordingly demonstrated that the turnarounds are actually single-motor switching between opposite-direction IFT trains rather than the behaviors of motors moving independently of IFT trains. We further improved the time resolution of single-motor imaging up to 30 ms to zoom into motor turnarounds, revealing diffusion during motor turnarounds, which unveils the mechanism of motor switching trains: detach-diffuse-attach. The subsequent single-molecule analysis of turnarounds unveiled location-dependent diffusion coefficients and diffusion times for both kinesin-2 and IFT-dynein motors. From correlating the diffusion times with IFT train frequencies, we estimated that kinesins tend to attach to the next train passing in the opposite direction. IFT-dynein, however, diffuses longer and lets one or two trains pass before attaching. This might be a direct consequence of the lower diffusion coefficient of the larger IFT-dynein. Our results provide important insights into how motors can cooperate to drive intracellular transport.

Anaplasma phagocytophilum Hijacks Flotillin and NPC1 Complex To Acquire Intracellular Cholesterol for Proliferation, Which Can Be Inhibited with Ezetimibe.

ABSTRACTThe intracellular cholesterol transport protein Niemann-Pick type C1 (NPC1) and lipid-raft protein flotillin (FLOT) are required for cholesterol uptake by the obligatory intracellular bacterium Anaplasma phagocytophilum and for infection, and each protein localizes to membrane-bound inclusions containing replicating bacteria. Here, we found striking localization of FLOT2 in NPC1-lined vesicles and a physical interaction between FLOT2 and NPC1. This interaction was cholesterol dependent, as a CRAC (cholesterol recognition/interaction amino acid cholesterol-binding) domain mutant of FLOT2 did not interact with NPC1, and the cholesterol-sequestering agent methyl-β-cyclodextrin reduced the interaction. The stomatin-prohibitin-flotillin-HflC/K domain of FLOT2, FLOT21–183, was sufficient for the unique FLOT2 localization and interaction with NPC1. NPC1, FLOT2, and FLOT21–183 trafficked to the lumen of Anaplasma inclusions. A loss-of-function mutant, NPC1P691S (mutation in the sterol-sensing domain), did not colocalize or interact with FLOT2 or with Anaplasma inclusions and inhibited infection. Ezetimibe is a drug that blocks cholesterol absorption in the small intestine by inhibiting plasma membrane Niemann-Pick C1-like 1 interaction with FLOTs. Ezetimibe blocked the interaction between NPC1 and FLOT2 and inhibited Anaplasma infection. Ezetimibe did not directly inhibit Anaplasma proliferation but inhibited host membrane lipid and cholesterol traffic to the bacteria in the inclusion. These data suggest that Anaplasma hijacks NPC1 vesicles containing cholesterol bound to FLOT2 to deliver cholesterol into Anaplasma inclusions to assimilate cholesterol for its proliferation. These results provide insights into mechanisms of intracellular cholesterol transport and a potential approach to inhibit Anaplasma infection by blocking cholesterol delivery into the lumen of bacterial inclusions.

Hydration Forces Dominate Surface Charge Dependent Lipid Bilayer Interactions under Physiological Conditions.

Lipid bilayer interactions are essential to a vast range of biological functions, such as intracellular transport mechanisms. Surface charging mediated by concentration dependent ion adsorption and desorption on lipid headgroups alters electric double layers as well as van der Waals and steric hydration forces of interacting bilayers. Here, we directly measure bilayer interactions during charge modulation in a symmetrically polarized electrochemical three-mirror interferometer surface forces apparatus. We quantify polarization and concentration dependent hydration and electric double layer forces due to cation adsorption/desorption. Our results demonstrate that exponential hydration layer interactions effectively describe surface potential dependent surface forces due to cation adsorption at high salt concentrations. Hence, electric double layers of lipid bilayers are exclusively dominated by inner Helmholtz charge regulation under physiological conditions. These results are important for rationalizing bilayer behavior under physiological conditions, where charge and concentration modulation may act as biological triggers for function and signaling.

Mechanisms of endocannabinoid transport in the brain

The endocannabinoids 2-arachidonoylglycerol (2-AG) and anandamide are among the best studied lipid messengers in the brain. By activating cannabinoid receptors in the CNS, endocannabinoids tune synaptic function, thereby influencing a variety of physiological and behavioural processes. Extensive research conducted over the last few decades has considerably enhanced our understanding of the molecular mechanisms and physiological functions of the endocannabinoid system. It is now well-established that endocannabinoids are synthesized by postsynaptic neurons and serve as retrograde messengers that suppress neurotransmitter release at central synapses. While the detailed mechanisms by which endocannabinoids gate synaptic function and behavioural processes are relatively well characterized, the mechanisms governing endocannabinoid transport at central synapses remain ill defined. Recently, several studies have begun to unravel the mechanisms governing intracellular and intercellular endocannabinoid transport. In this review, we will focus on new advances in the mechanisms of intracellular and synaptic endocannabinoid transport in the CNS. LINKED ARTICLES: This article is part of a themed issue on New discoveries and perspectives in mental and pain disorders. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v179.17/issuetoc.