Cytoplasmic Trafficking Research Articles

Epithelial cancer cells exhibit different electrical properties when cultured in 2D and 3D environments

BackgroundMany drug development and toxicology studies are performed using cells grown in monolayers in well-plates and flasks, despite the fact that these are widely held to be different to cells found in the native environment. 3D, tissue engineered, organotypical tissue culture systems have been developed to be more representative of the native tissue environment than standard monolayer cultures. Whilst the biochemical differences between cells grown in 2D and 3D culture have been explored, the changes on the electrophysiological properties of the cells have not. MethodsWe compared the electrophysiological properties of primary normal oral keratinocytes (nOK) and cancerous abnormal oral keratinocytes (aOK), cultured in standard monolayer and reconstituted 3D organotypical tissue cultures. The electrophysiological properties of populations of the cells were analysed using dielectrophoresis. The intracellular conductivity of aOK was significantly increased when grown in organotypical cultures compared to counterpart cells grown in monolayer cultures. Results3D cultured aOK showed almost identical intracellular conductivity to nOK also grown in organotypical cultures, but significantly different to aOK grown in monolayers. The effective membrane capacitance of aOK grown in 3D was found to be significantly higher than nOK, but there was no significant difference between the electrophysiological properties of nOK grown in 2D and 3D cultures. General significanceThis work suggests that factors such as cell shape and cytoplasmic trafficking between cells play an important role in their electrophysiology, and highlights the need to use in vitro models more representative of native tissue when studying cell electrophysiological properties.

Abstract SY29-03: Following single mRNAs from birth to death in living cells and tissues.

Abstract Imaging has been an essential tool to analyze the dynamic properties of RNA. New technologies in optical microscopy and tagging methods have allowed us to detect and track individual mRNA molecules in single living cells, yielding insights that could not have been obtained through any ensemble measurement: for instance in measuring the time for transcription elongation or nuclear pore transport. We have been dedicated to developing and implementing these technologies to further the understanding of dynamics of mRNA regulation in cells. We have utilized various microscopic approaches to analyze the components of real-time transcription of endogenous genes in living tissues of a transgenic mouse where the mRNAs are labeled by a fluorescent RNA binding protein. These approaches employ a plethora of imaging techniques, ranging from confocal and multiphoton microscopy, long-term cell imaging, high-speed real-time widefield microscopy, single molecule tracking, super-registration microscopy and fluorescence fluctuation analysis. The kinetics of the key elements of RNA metabolism: initiation, elongation, termination, as well as nuclear pore export, cytoplasmic trafficking, localization, translation and decay are now tractable at the single molecule level in vivo. By using mathematical modeling to extract the quantitative parameters of these kinetics, we can describe these processes precisely in living cells, leading to an understanding of the regulatory events governing gene expression-fundamental to the mechanism of disease. Supported by NIH GMS Grants to RH Singer. Citation Format: Robert Singer. Following single mRNAs from birth to death in living cells and tissues. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr SY29-03. doi:10.1158/1538-7445.AM2013-SY29-03

Nucleozin Targets Cytoplasmic Trafficking of Viral Ribonucleoprotein-Rab11 Complexes in Influenza A Virus Infection

Novel antivirals are needed to supplement existing control strategies for influenza A virus (IAV). A promising new class of drug, exemplified by the compound nucleozin, has recently been identified that targets the viral nucleoprotein (NP). These inhibitors are thought to act as "molecular staples" that stabilize interactions between NP monomers, promoting the formation of nonfunctional aggregates. Here we detail the inhibitory mechanism of nucleozin, finding that the drug has both early- and late-acting effects on the IAV life cycle. When present at the start of infection, it inhibited viral RNA and protein synthesis. However, when added at later time points, it still potently blocked the production of infectious progeny but without affecting viral macromolecular synthesis. Instead, nucleozin blocked the cytoplasmic trafficking of ribonucleoproteins (RNPs) that had undergone nuclear export, promoting the formation of large perinuclear aggregates of RNPs along with cellular Rab11. This effect led to the production of much reduced amounts of often markedly smaller virus particles. We conclude that the primary target of nucleozin is the viral RNP, not NP, and this work also provides proof of the principle that IAV replication can be effectively inhibited by blocking cytoplasmic trafficking of the viral genome.

Novel <i>SCN3B</i> Mutation Associated With Brugada Syndrome Affects Intracellular Trafficking and Function of Nav1.5

Brugada syndrome (BrS) is characterized by specific alterations on ECG in the right precordial leads and associated with ventricular arrhythmia that may manifest as syncope or sudden cardiac death. The major causes of BrS are mutations in SCN5A for a large subunit of the sodium channel, Nav1.5, but a mutation in SCN3B for a small subunit of sodium channel, Navβ3, has been recently reported in an American patient. A total of 181 unrelated BrS patients, 178 Japanese and 3 Koreans, who had no mutations in SCN5A, were examined for mutations in SCN3B by direct sequencing of all exons and adjacent introns. A mutation, Val110Ile, was identified in 3 of 178 (1.7%) Japanese patients, but was not found in 480 Japanese controls. The SCN3B mutation impaired the cytoplasmic trafficking of Nav1.5, the cell surface expression of which was decreased in transfected cells. Whole-cell patch clamp recordings of the transfected cells revealed that the sodium currents were significantly reduced by the SCN3B mutation. The Val110Ile mutation of SCN3B is a relatively common cause of SCN5A-negative BrS in Japan, which has a reduced sodium current because of the loss of cell surface expression of Nav1.5.

Highly acetylated tubulin permits enhanced interactions with and trafficking of plasmids along microtubules

Microtubule-based transport is required for plasmid translocation to the nucleus during transfections, and having stable structures could enhance this movement. In previous studies in which the cytoskeleton was disrupted, we found that populations of microtubules remain that are stable and highly acetylated. By increasing the levels of acetylated tubulin through inhibition of the tubulin deacetylase HDAC6, we observe more rapid plasmid nuclear localization of transfected plasmids and greater levels of gene transfer. In this study, we sought to understand plasmid movement in cells with enhanced tubulin acetylation. Using variations of a microtubule spin down assay, we found that plasmids bound to hyper-acetylated microtubules to a greater degree than they did to unmodified microtubules. To determine if microtubule acetylation also affects cytoplasmic trafficking, plasmid movement was evaluated in real time by particle tracking in cells with varying levels of acetylated microtubules. We found that plasmids display greater net rates of movement, spend more time in productive motion and display longer runs of continuous motion in cells with highly acetylated microtubules compared to those with fewer modifications. These results all suggest that plasmid movement is enhanced along highly acetylated microtubules, reducing the time spent in the cytoplasm prior to nuclear import. Taken together, these findings provide a foundation for determining how modulation of microtubule acetylation can be used as a means to increase intracellular trafficking of plasmids and enhance gene therapy.

Cytoplasmic Trafficking, Endosomal Escape, and Perinuclear Accumulation of Adeno-Associated Virus Type 2 Particles Are Facilitated by Microtubule Network

Understanding adeno-associated virus (AAV) trafficking is critical to advance our knowledge of AAV biology and exploit novel aspects of vector development. Similar to the case for most DNA viruses, after receptor binding and entry, AAV traverses the cytoplasm and deposits the viral genome in the cell nucleus. In this study, we examined the role of the microtubule (MT) network in productive AAV infection. Using pharmacological reagents (e.g., nocodazole), live-cell imaging, and flow cytometry analysis, we demonstrated that AAV type 2 (AAV2) transduction was reduced by at least 2-fold in the absence of the MT network. Cell surface attachment and viral internalization were not dependent on an intact MT network. In treated cells at 2 h postinfection, quantitative three-dimensional (3D) microscopy determined a reproducible difference in number of intracellular particles associated with the nuclear membrane or the nucleus compared to that for controls (6 to 7% versus 26 to 30%, respectively). Confocal microscopy analysis demonstrated a direct association of virions with MTs, further supporting a critical role in AAV infection. To investigate the underling mechanisms, we employed single-particle tracking (SPT) to monitor the viral movement in real time. Surprisingly, unlike other DNA viruses (e.g., adenovirus [Ad] and herpes simplex virus [HSV]) that display bidirectional motion on MTs, AAV2 displays only unidirectional movement on MTs toward the nuclei, with peak instantaneous velocities at 1.5 to 3.5 μm/s. This rapid and unidirectional motion on MTs lasts for about 5 to 10 s and results in AAV particles migrating more than 10 μm in the cytoplasm reaching the nucleus very efficiently. Furthermore, electron microscopy analysis determined that, unlike Ad and HSV, AAV2 particles were transported on MTs within membranous compartments, and surprisingly, the acidification of AAV2-containing endosomes was delayed by the disruption of MTs. These findings together suggest an as-yet-undescribed model in which after internalization, AAV2 exploits MTs for rapid cytoplasmic trafficking in endosomal compartments unidirectionally toward the perinuclear region, where most acidification events for viral escape take place.

Intrinsic and extrinsic negative regulators of nuclear protein transport processes

The nuclear–cytoplasmic protein transport is a critical process in cellular events. The identification of transport signals (nuclear localization signal and nuclear export signal) and their receptors has facilitated our understanding of this expanding field. Nuclear transport must be appropriately regulated to deliver proteins through the nuclear pore when their functions are required in the nucleus, and to export them into the cytoplasm when they are not needed in the nucleus. Altered nuclear transport processes have been observed in stressed cells, which would change gene expressions. Some viruses interfere with nuclear transport in host cells to evade immune defense. Moreover, certain transport factors negatively regulate nuclear protein transport in cells. Understanding the regulatory mechanisms of nuclear–cytoplasmic trafficking not only provides important information about cellular processes, but also is of use for developing specific inhibitors for transport pathways.

The mitotic spindle protein SPAG5/Astrin connects to the Usher protein network postmitotically

BackgroundMutations in the gene for Usher syndrome 2A (USH2A) are causative for non-syndromic retinitis pigmentosa and Usher syndrome, a condition that is the most common cause of combined deaf-blindness. To gain insight into the molecular pathology underlying USH2A-associated retinal degeneration, we aimed to identify interacting proteins of USH2A isoform B (USH2AisoB) in the retina.ResultsWe identified the centrosomal and microtubule-associated protein sperm-associated antigen (SPAG)5 in the retina. SPAG5 was also found to interact with another previously described USH2AisoB interaction partner: the centrosomal ninein-like protein NINLisoB. Using In situ hybridization, we found that Spag5 was widely expressed during murine embryonic development, with prominent signals in the eye, cochlea, brain, kidney and liver. SPAG5 expression in adult human tissues was detected by quantitative PCR, which identified expression in the retina, brain, intestine, kidney and testis. In the retina, Spag5, Ush2aisoB and NinlisoB were present at several subcellular structures of photoreceptor cells, and colocalized at the basal bodies.ConclusionsBased on these results and on the suggested roles for USH proteins in vesicle transport and providing structural support to both the inner ear and the retina, we hypothesize that SPAG5, USH2AisoB and NINLisoB may function together in microtubule-based cytoplasmic trafficking of proteins that are essential for cilium formation, maintenance and/or function.

Following Single mRNAs from Birth to Death in Living Cells

Live cell imaging has been instrumental in analyzing the dynamic properties of RNA. New technologies in optical microscopy and fluorescent probe development have been pushing the envelope of our analysis capabilities. We have been dedicated to developing and implementing these technologies to further the understanding of single mRNA dynamics in cells and organisms. We have utilized computational approaches to analyze real-time transcription activities of endogenous genes from yeast to human cells. We have employed a plethora of imaging methods, ranging from confocal and multiphoton microscopy, long-term cell imaging, high-speed real-time widefield microscopy, single molecule tracking, and we have developed super-registration microscopy and fluorescence fluctuation analysis. We have investigated key processes of RNA synthesis: initiation, elongation, termination, as well as nuclear pore export, cytoplasmic trafficking, localization and decay. Mathematical modeling allowed us to extract quantitative kinetic parameters that precisely describe these processes in living cells.Supported by NIH GMS Grants to RH Singer.

Cytoplasmic Trafficking, Endosomal Escape, and Perinuclear Accumulation of Adeno-Associated Virus Type 2 Particles Are Facilitated by Microtubule Network

Cytoplasmic Trafficking, Endosomal Escape, and Perinuclear Accumulation of Adeno-Associated Virus Type 2 Particles Are Facilitated by Microtubule Network

The mRNA Stability Factor HuR Inhibits MicroRNA-16 Targeting of COX-2

Commonly observed in colorectal cancer is the elevated expression of the prostaglandin (PG) synthase COX-2. In normal intestinal epithelium, the COX-2 mRNA is targeted for rapid decay through the 3'-untranslated region (3'-UTR) adenylate- and uridylate (AU)-rich element (ARE), whereas in tumors ARE-mediated decay is compromised. Here we show that the COX-2 ARE can mediate degradation through microRNA (miRNA)-mediated regulation. We identified miR-16 to bind the COX-2 3'-UTR and inhibit COX-2 expression by promoting rapid mRNA decay. In colorectal cancer cells and tumors, miR-16 levels were decreased approximately twofold and miR-16 expression in cancer cells attenuated COX-2 expression and PG synthesis. The COX-2 ARE is also bound by the RNA-binding protein HuR. In colorectal cancer tumors, HuR is overexpressed and localized within the cytoplasm, where it promotes ARE-mRNA stabilization. Under conditions of HuR overexpression, miR-16 was unable to promote rapid mRNA decay through the COX-2 ARE. Ribonucleoprotein immunoprecipitation of HuR showed direct association with miR-16 that was reversed when cytoplasmic trafficking of HuR was inhibited. Furthermore, this interaction between HuR and miR-16 promoted the downregulation of miR-16. These new results identify miR-16 as a central posttranscriptional regulator of COX-2 and show the ability of elevated levels of HuR to antagonize miR-16 function. Along with insight into altered ARE-mediated mRNA decay observed in colorectal cancer, these findings provide a new explanation for tumor-derived loss of miR-16.

The cell biology of TRIM5α.

The tripartite motif (TRIM)-containing proteins are involved in many cellular functions such as cell signaling, apoptosis, cell differentiation, and immune modulation. TRIM5 proteins, including TRIM5α and TRIM-Cyp, are known to possess antiretroviral activity against many different retroviruses. Besides being retroviral restriction factors, TRIM5 proteins participate in other cellular functions that have recently emerged in the study of TRIM5α. In this review, we discuss properties of TRIM5α such as cytoplasmic body formation, protein turnover, and trafficking. Also, we discuss recent insights into innate immune modulation mediated by TRIM5α, highlighting the various functions TRIM5α has in cellular processes.

Human Immunodeficiency Virus Rev-Binding Protein Is Essential for Influenza A Virus Replication and Promotes Genome Trafficking in Late-Stage Infection

Influenza A virus uses cellular protein transport systems (e.g., CRM1-mediated nuclear export and Rab11-dependent recycling endosomes) for genome trafficking from the nucleus to the plasma membrane, where new virions are assembled. However, the detailed mechanisms of these events have not been completely resolved, and additional cellular factors are probably required. Here, we investigated the role of the cellular human immunodeficiency virus (HIV) Rev-binding protein (HRB), which interacts with influenza virus nuclear export protein (NEP), during the influenza virus life cycle. By using small interfering RNAs (siRNAs) and overexpression of a dominant negative HRB protein fragment, we show that cells lacking functional HRB have significantly reduced production of influenza virus progeny and that this defect results from impaired viral ribonucleoprotein (vRNP) delivery to the plasma membrane in late-stage infection. Since HRB colocalizes with influenza vRNPs early after their delivery to the cytoplasm, it may mediate a connection between the nucleocytoplasmic transport machinery and the endosomal system, thus facilitating the transfer of vRNPs from nuclear export to cytoplasmic trafficking complexes. We also found an association between NEP and HRB in the perinuclear region, suggesting that NEP may contribute to this process. Our results identify HRB as a second endosomal factor with a crucial role in influenza virus genome trafficking, suggest cooperation between unique endosomal compartments in the late steps of the influenza virus life cycle, and provide a common link between the cytoplasmic trafficking mechanisms of influenza virus and HIV.

Transcription factor plasmid binding modulates microtubule interactions and intracellular trafficking during gene transfer

For non-viral gene delivery to be successful, plasmids must move through the cytoplasm to the nucleus in order to be transcribed. While the cytoskeletal meshwork acts as a barrier to plasmid DNA movement in the cytoplasm, the microtubule network is required for directed plasmid trafficking to the nucleus. We have shown previously that plasmid-microtubule interactions require cytoplasmic adapter proteins such as molecular motors, transcription factors (TFs) and importins. However, not all plasmid sequences support these interactions to allow movement to the nucleus. We now demonstrate that microtubule-DNA interactions can show sequence specificity with promoters containing binding sites for cyclic AMP response-element binding protein (CREB), including the cytomegalovirus immediate early promoter (CMV(iep)). Plasmids containing CREB-binding sites showed stringent interactions in an in vitro microtubule-binding assay. Using microinjection and real-time particle tracking, we show that the inclusion of TF binding sites within plasmids permits cytoplasmic trafficking of plasmids during gene transfer. We found that CREB-binding sites are bound by CREB in the cytoplasm during transfection, and allow for enhanced rates of movement and subsequent nuclear accumulation. Moreover, small interfering RNA knockdown of CREB prevented this enhanced trafficking. Therefore, TF binding sites within plasmids are necessary for interactions with microtubules and enhance movement to the nucleus.

Differential effect of acute and persistent Junín virus infections on the nucleo-cytoplasmic trafficking and expression of heterogeneous nuclear ribonucleoproteins type A and B

Heterogeneous nuclear ribonucleoproteins A and B (hnRNPs A/B), cellular RNA-binding proteins that participate in splicing, trafficking, translation and turnover of mRNAs, have been implicated in the life cycles of several cytoplasmic RNA viruses. Here, we demonstrate that silencing of hnRNPs A1 and A2 significantly reduces the replication of the arenavirus Junín virus (JUNV), the aetiological agent of Argentine haemorrhagic fever. While acute JUNV infection did not modify total levels of expression of hnRNPs A/B in comparison with uninfected cells, non-cytopathic persistent infection exhibited low levels of these cell proteins. Furthermore, acutely infected cells showed a cytoplasmic relocalization of overexpressed hnRNP A1, probably related to the involvement of this protein in virus replicative cycle. This cytoplasmic accumulation was also observed in cells expressing viral nucleoprotein (N), and co-immunoprecipitation studies revealed the interaction between hnRNP A1 and N protein. By contrast, a predominantly nuclear distribution of overexpressed hnRNP A1 was found during persistent infection, even in the presence of endogenous or overexpressed N protein, indicating a differential modulation of nucleo-cytoplasmic trafficking in acute and persistent JUNV infections.

Proteomic analysis of urinary exosomes from patients of early IgA nephropathy and thin basement membrane nephropathy

To identify biomarker candidates associated with early IgA nephropathy (IgAN) and thin basement membrane nephropathy (TBMN), the most common causes presenting isolated hematuria in childhood, a proteomic approach of urinary exosomes from early IgAN and TBMN patients was introduced. The proteomic results from the patients were compared with a normal group to understand the pathophysiological processes associated with these diseases at the protein level. The urinary exosomes, which reflect pathophysiological processes, collected from three groups of young adults (early IgAN, TBMN, and normal) were trypsin-digested using a gel-assisted protocol, and quantified by label-free LC-MS/MS, using an MS(E) mode. A total of 1877 urinary exosome proteins, including cytoplasmic, membrane, and vesicle trafficking proteins, were identified. Among the differentially expressed proteins, four proteins (aminopeptidase N, vasorin precursor, α-1-antitrypsin, and ceruloplasmin) were selected as biomarker candidates to differentiate early IgAN from TBMN. We confirmed the protein levels of the four biomarker candidates by semi-quantitative immunoblot analysis in urinary exosomes independently prepared from other patients, including older adult groups. Further clinical studies are needed to investigate the diagnostic and prognostic value of these urinary markers for early IgAN and TBMN. Taken together, this study showed the possibility of identifying biomarker candidates for human urinary diseases using urinary exosomes and might help to understand the pathophysiology of early IgAN and TBMN at the protein level.

Abstract 5416: The low-molecular-weight compound MS-444 selectively targets cancer cells via inhibition of the mRNA stability factor HuR

Abstract Colorectal cancer (CRC) is the third most common cancer among American adults and a leading cause of cancer-related mortality. The initiation and progression of colorectal tumorigenesis is characterized by genetic alterations that promote aberrant expression of oncogenic and tumor-promoting factors. In normal cells, the mRNAs of these inflammation- and cancer-associated genes are targeted for rapid decay through AU-rich elements (ARE) present in the mRNA 3’UTR. However, loss of ARE-mediated mRNA decay is a characteristic feature contributing to pathogenic gene overexpression in colorectal cancer. Our prior work identified enhanced expression of the RNA-binding protein HuR to occur during colon tumorigenesis and HuR overexpression is correlated with poor clinical prognosis. In this capacity, HuR acts as a mRNA stability factor by promoting ARE-containing mRNA stabilization. Therefore, HuR has emerged as a novel pharmacological target with potential selectivity for cancer cells. This was examined by treating HuR-overexpressing CRC cell lines HCT116, SW480, RKO, and HT-29 with the small molecule inhibitor of HuR MS-444. Growth inhibition was observed in all CRC lines with IC50 values of 7-12 µM and cancer cell selectivity was observed with 4- to 5-fold increased MS-444 IC50 in non-transformed intestinal and colonic epithelial cells. HuR is predominantly nuclear in non-transformed cells, whereas in CRC cells aberrant cytoplasmic HuR trafficking is observed indicating that cytoplasmic localization is a necessary component for HuR-mediated ARE-mRNA stabilization and cancer cell survival. In HCT116 cells treated with MS-444, complete inhibition of cytoplasmic HuR was observed along with induction of apoptosis. Similarly, long-term siRNA-mediated knockdown of HuR in HCT116 cells resulted in cellular apoptosis, and cells with attenuated levels of HuR exhibited a 5-fold higher sensitivity to MS-444 treatment. Expression profiling revealed increased expression of many pro-apoptotic proteins to be present in colon cancer cells resulting from MS-444 treatment. The expression of Fas ligand, a TNF superfamily member, is significantly increased, accompanied by elevated expression of caspase 8 and cell death effectors such as Bax and Bid. These results indicate that MS-444 treatment triggers a Fas-dependent signaling cascade mediated through cellular caspases, thereby resulting in cell death. Taken together, these findings indicate that small molecule-based inhibition of HuR results in selective cancer cell death while also indicating that overexpression of HuR is a necessary component for colorectal tumor cell survival. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5416. doi:10.1158/1538-7445.AM2011-5416

The Dynactin Complex Enhances the Speed of Microtubule-Dependent Motions of Adenovirus Both Towards and Away from the Nucleus

Unlike transport vesicles or organelles, human adenovirus (HAdV) directly binds to the microtubule minus end-directed motor dynein for transport to the nucleus. The dynein cofactor dynactin enhances nuclear transport of HAdV and boosts infection. To determine if dynactin has a specific role in cytoplasmic trafficking of incoming HAdV on microtubules, we used live cell spinning disc confocal microscopy at 25 Hz acquisition frequency and automated tracking of single virus particles at 20–50 nm spatial resolution. Computational dissection by machine-learning algorithms extracted specific motion patterns of viral trajectories. We found that unperturbed cells supported two kinds of microtubule-dependent motions, directed motions (DM) and fast drifts (FD). DM had speeds of 0.2 to 2 μm/s and run lengths of 0.4 up to 7 μm, while FD were slower and less extensive at 0.02 to 0.4 μm/s and 0.05 to 2.5 μm. Dynactin interference by overexpression of p50/dynamitin or a coiled-coil domain of p150/Glued reduced the speeds and amounts of both center- and periphery-directed DM but not FD, and inhibited infection. These results indicate that dynactin enhances adenovirus infection by increasing the speed and efficiency of dynein-mediated virus motion to the nucleus, and, surprisingly, also supports a hereto unknown motor activity for virus transport to the cell periphery.

A Rab11- and Microtubule-Dependent Mechanism for Cytoplasmic Transport of Influenza A Virus Viral RNA

The viral RNA (vRNA) genome of influenza A virus is replicated in the nucleus, exported to the cytoplasm as ribonucleoproteins (RNPs), and trafficked to the plasma membrane through uncertain means. Using fluorescent in situ hybridization to detect vRNA as well as the live cell imaging of fluorescently labeled RNPs, we show that an early event in vRNA cytoplasmic trafficking involves accumulation near the microtubule organizing center in multiple cell types and viral strains. Here, RNPs colocalized with Rab11, a pericentriolar recycling endosome marker. Cytoplasmic RNP localization was perturbed by inhibitors of vesicular trafficking, microtubules, or the short interfering RNA-mediated depletion of Rab11. Green fluorescent protein (GFP)-tagged RNPs in living cells demonstrated rapid, bidirectional, and saltatory movement, which is characteristic of microtubule-based transport, and also cotrafficked with fluorescent Rab11. Coprecipitation experiments showed an interaction between RNPs and the GTP-bound form of Rab11, potentially mediated via the PB2 subunit of the polymerase. We propose that influenza virus RNPs are routed from the nucleus to the pericentriolar recycling endosome (RE), where they access a Rab11-dependent vesicular transport pathway to the cell periphery.

Special Section on Controlled Drug Delivery

Controlled drug delivery has been attracting a great deal of attention in the medical community for years as an efficient way of providing treatment for a wide class of diseases. The common principle on which various drug delivery devices are based is mass transfer of the given drug toward particular organs, in which either the mass transfer rate, or place, or both are prescribed according to certain medical protocols. Much progress has been achieved in the design and development of various controlled drug delivery systems, and many people routinely take medicine designed for controlled release. Mathematical modeling of drug delivery systems is very important because a successful model can provide a better understanding and a quantitative description of the physical, chemical and biological processes governing the performance of the systems. On the basis of this description, better controlled drug delivery systems can be designed. We hope that this collection of papers will result in an increased attention of applied mathematicians to this class of important mass transfer and control problems. We also hope that practitioners will become more aware of the mathematical results that can be useful. The papers differ in mathematical sophistication, ranging from models described by systems of ODEs to quite complex PDE problems. They consider quite a few different aspects of drug delivery thus introducing a significant portion of the field. Each paper has an extensive introduction that should make the paper understandable to an applied mathematician who is not an expert in drug delivery. A discussion of controlled drug delivery in cancer immunotherapy is presented in “Controlled Drug Delivery in Cancer Immunotherapy: Stability, Optimization, and Monte Carlo Analysis" by Minelli, Topputo, and Bernelli-Zazzera, who formulate and solve an optimal control problem and show that the control policy is effective even when the patient's initial conditions are uncertain. A hybrid model of cell population dynamics, where cells are discrete elements whose dynamics depend on continuous intracellular and extracellular processes, is developed in “Hybrid Model of Erythropoiesis and Leukemia Treatment with Cytosine Arabinoside" by Kurbatova, Bernard, Bessonov, Crauste, Demin, Dumontet, Fischer, and Volpert, to simulate the evolution of immature red blood cells in the bone marrow. The model is used to study normal and leukemic red blood cell production and treatment of leukemia. In “Quadratic Models to Fit Experimental Data of Paclitaxel Release Kinetics from Biodegradable Polymers," Blanchet, Delfour, and Garon validate three ODE models against experimental data in order to better understand drug release kinetics. A model for drug diffusion from a spherical polymeric drug delivery device is considered in “Asymptotic and Numerical Results for a Model of Solvent-Dependent Drug Diffusion through Polymeric Spheres" by McCue, Hsieh, Moroney, and Nelson. Here the solvent diffuses into the polymer, which transitions from a glassy to a rubbery state, thus resulting in a moving boundary problem. In “Model Reduction Strategies Enable Computational Analysis of Controlled Drug Release from Cardiovascular Stents," D'Angelo, Zunino, Porpora, Morlacchi, and Migliavacca deal with drug-eluting stents and consider a hierarchy of mathematical models ranging from a lumped ODE model to fully three-dimensional models for drug transfer in the artery. A gene delivery of nucleic acid to the cell nucleus problem is discussed in “Modeling the Early Steps of Cytoplasmic Trafficking in Viral Infection and Gene Delivery," in which Amoruso, Lagache, and Holcman focus on plasmid DNA and virus cytoplasmic trafficking. The editors thank all the authors and reviewers for their contributions to this collection.