Microtubule-dependent Process Research Articles

Myosin-5a facilitates stress granule formation by interacting with G3BP1

Stress granules (SGs) are non-membranous organelles composed of mRNA and proteins that assemble in the cytosol when the cell is under stress. Although the composition of mammalian SGs is both cell-type and stress-dependent, they consistently contain core components, such as Ras GTPase activating protein SH3 domain binding protein 1 (G3BP1). Upon stress, living cells rapidly assemble micrometric SGs, sometimes within a few minutes, suggesting that SG components may be actively transported by the microtubule and/or actin cytoskeleton. Indeed, SG assembly has been shown to depend on the microtubule cytoskeleton and the associated motor proteins. However, the role of the actin cytoskeleton and associated myosin motor proteins remains controversial. Here, we identified G3BP1 as a novel binding protein of unconventional myosin-5a (Myo5a). G3BP1 uses its C-terminal RNA-binding domain to interact with the middle portion of Myo5a tail domain (Myo5a-MTD). Suppressing Myo5a function in mammalian cells, either by overexpressing Myo5a-MTD, eliminating Myo5a gene expression, or treatment with myosin-5 inhibitor, inhibits the arsenite-induced formation of both small and large SGs. This is different from the effect of microtubule disruption, which abolishes the formation of large SGs but enhances the formation of small SGs under stress conditions. We therefore propose that, under stress conditions, Myo5a facilitates the formation of SGs at an earlier stage than the microtubule-dependent process.

FER regulates endosomal recycling and is a predictor for adjuvant taxane benefit in breast cancer.

Elevated expression of non-receptor tyrosine kinase FER is an independent prognosticator that correlates with poor survival of high-grade and basal/triple-negative breast cancer (TNBC) patients. Here, we show that high FER levels are also associated with improved outcomes after adjuvant taxane-based combination chemotherapy in high-risk, HER2-negative patients. In TNBC cells, we observe a causal relation between high FER levels and sensitivity to taxanes. Proteomics and mechanistic studies demonstrate that FER regulates endosomal recycling, a microtubule-dependent process that underpins breast cancer cell invasion. Using chemical genetics, we identify DCTN2 as a FER substrate. Our work indicates that the DCTN2 tyrosine 6 is essential for the development of tubular recycling domains in early endosomes and subsequent propagation of TNBC cell invasion in 3D. In conclusion, we show that high FER expression promotes endosomal recycling and represents a candidate predictive marker for the benefit of adjuvant taxane-containing chemotherapy in high-risk patients, including TNBC patients.

Abstract LB203: FER regulates endosomal recycling and is a candidate predictor for taxane benefit in breast cancer

Abstract To this day, targeted treatment options for patients with triple negative metastatic breast cancer (TNBC) are still virtually absent. Previously, we showed that elevated expression of the tyrosine kinase FER is an independent prognosticator that correlates with poor survival of high-grade and basal/TNBC patients. Here, we show that high FER levels are also associated with improved outcome after adjuvant taxane-based combination chemotherapy, in high-risk, HER2-negative patients. In TNBC cells, we observed a causal relation between high FER levels and sensitivity to taxanes. Our proteomics and mechanistic studies demonstrated that FER regulates endosomal recycling, a microtubule-dependent process that underpins breast cancer cell invasion. Using chemical genetics, we identified DCTN2 as a novel FER substrate. Our work indicates that the DCTN2 tyrosine 6 is essential for tubular recycling domains development in early endosomes and subsequent propagation of TNBC cell invasion in 3D. In conclusion, we show that high FER expression promotes endosomal recycling and represents a candidate predictive marker for benefit of adjuvant taxane-containing chemotherapy in high-risk patients, including TNBC patients. Citation Format: Sandra Tavares, Nalan Liv, Milena Pasolli, Mark Opdam, Max Ratze, Manuel Saornil, Lilian Sluimer, Rutger Hengeveld, Robert van Es, Erik van Werkhoven, Harmjan Vos, Holger Rehmann, Boudewijn Burgering, Hendrika Oosterkamp, Susanne Lens, Judith Klumperman, Sabine Linn, Patrick Derksen. FER regulates endosomal recycling and is a candidate predictor for taxane benefit in breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr LB203.

Monitoring of the Cytoskeleton-Dependent Intracellular Trafficking of Fluorescent Iron Oxide Nanoparticles by Nanoparticle Pulse-Chase Experiments in C6 Glioma Cells.

Iron oxide nanoparticles (IONPs) are used for various biomedical and therapeutic approaches. To investigate the uptake and the intracellular trafficking of IONPs in neural cells we have performed nanoparticle pulse-chase experiments to visualize the internalization and the fate of fluorescent IONPs in C6 glioma cells and astrocyte cultures. Already a short exposure to IONPs for 10min at 4°C (nanoparticle pulse) allowed binding of substantial amounts of nanoparticles to the cells, while internalization of IONPs into the cell was prevented. The uptake of bound IONPs and the intracellular trafficking was started by increasing the temperature to 37°C (chase period). While hardly any cellular fluorescence nor any iron staining was detectable directly after the nanoparticle pulse, dotted cellular fluorescence and iron patterns appeared already within a few minutes after start of the chase incubation and became intensified in the perinuclear region during further incubation for up to 90min. Longer chase incubations resulted in separation of the fluorescent coat from the core of the internalized IONPs. Disruption of actin filaments in C6 cells strongly impaired the internalization of IONPs, whereas destabilization of microtubules traped IONP-containing vesicles to the plasma membrane. In conclusion, nanoparticle pulse-chase experiments allowed to synchronize the cellular uptake of fluorescent IONPs and to identify for C6 cells an actin-dependent early and a microtubule-dependent later process in the intracellular trafficking of fluorescent IONPs.

Prix Master 2016 de la SFM

In the normal course of muscle fiber development, myonuclei actively position and adapt a precise localization in mature fibers, shaping MyoNuclear Domains (MNDs). Myonuclei positioning in fibers appears to be essential for muscle function as defects in MNDs settings are always associated with dysfunction (i.e., centronuclear myopathy, sarcopenia). Previous studies have shown that myonuclei positioning in fibers is reversible, suggesting that in pathologies presenting MNDs impairment, myonuclei could be re-addressed to the "correct" position in fibers and this could benefit to muscle function. Cytoskeleton networks, and particularly microtubules, have been implicated in early nuclei localization in myotubes. As the microtubule network is completely redesigned during muscle maturation, we hypothesized that "microtubules associated proteomes" would change between immature and mature fibers and contribute to a microtubule-dependent process resulting in MNDs setting and maintenance in mature fibers. We performed an in vitro biochemical approach to isolate microtubules partners in immature (myotubes) and mature myofibers. Using mass-spectrometry identification, we selected 244 candidates, differentially associated/expressed with microtubules during myofiber maturation and potentially controlling MNDs settings. We are currently conducting a siRNA screen approach on these candidates to decipher their respective implication in early and late phases of MNDs establishment, using an unbiased assay developed by our team allowing statistical analysis of MNDs regarding myonuclei content. This approach will lead to the identification of new pathways related to nuclear positioning and MNDs setting in normal condition and in myopathies associated to MNDs impairment such as CNMs.

Keratin 8/18 regulation of insulin receptor signaling and trafficking in hepatocytes through a concerted phosphoinositide-dependent Akt and Rab5 modulation.

Keratins (Ks) are epithelial cell intermediate filament (IF) proteins that are expressed as pairs in a differentiation-regulated manner. Hepatocyte IFs are made only of K8/K18 pairs, which means that a K8 loss in K8-null mice leads to degradation of K18. Functionally, there is accumulating evidence that IFs contribute to signaling platforms. Here, we investigate the role of K8/K18 IFs in the regulation of insulin receptor (IR) signaling and trafficking in hepatocytes. We find that the IR substrate 1 (IRS1)/PI3K/Akt signaling cascade-downstream of IR-displays prolonged activation in K8-null compared with wild-type hepatocytes. Assessment of the Akt/mammalian target of rapamycin complex 1-mediated feedback loop to IRS1/PI3K, in the absence or presence of drug inhibitors, further supports a preferential K8/K18 IF intervention at the surface membrane. In K8-null hepatocytes, IR trafficking vesicles that are labeled by Rab5/EEA1/phosphatidylinositol 3-phosphate accumulate at a juxtanuclear region via a microtubule-dependent process. Moreover, interference with phosphatidylinositol 4,5-biphosphate signaling aggravates IR/Rab5 accumulation. Overall, results uncover K8/K18 IF regulation of IR signaling via a concerted modulation of phosphatidylinositol 4,5-biphosphate-dependent IRS1/PI3K/Akt signaling and Rab5/phosphatidylinositol 3-phosphate/microtubule trafficking in hepatocytes.-Roux, A., Loranger, A., Lavoie, J. N., Marceau, N. Keratin 8/18 regulation of insulin receptor signaling and trafficking in hepatocytes through a concerted phosphoinositide-dependent Akt and Rab5 modulation.

Abstract P4-04-04: Eribulin impairs TGF-β type I receptor localization and signaling in BT-549 cells

Abstract Microtubule targeting agents (MTAs) continue to be some of the most valuable drugs used to treat breast cancer. While decades of research have shown that these drugs cause mitotic arrest in cells by suppressing the dynamic instability of microtubules, recent evidence demonstrates that the ability of MTAs to disrupt microtubule-dependent transport of key signaling components, including proteins and microRNAs, in interphase cells likely contributes to their anticancer actions. TGF-β signaling is a driver of oncogenesis and epithelial-to-mesenchymal transition (EMT) that involves multiple protein trafficking and intracellular signaling events. Ligand mediated activation of the cell surface TGF-β receptors leads to downstream signaling in the canonical and the non-canonical pathways that collectively lead to the expression of proteins implicated in EMT, including Snail and Slug. Additionally, TGF-β type 1 receptor (TGF-βR1) undergoes constant cycling from the plasma membrane to the cytosol, a microtubule-dependent process. We tested the hypothesis that a short-term treatment of breast cancer cells with eribulin or 3 other clinically relevant MTAs would differentially disrupt interphase microtubules and alter the transport and downstream signaling of TGF-βR1. BT-549 cells were treated for 2 h with concentrations of MTAs that cause comparable disruption of the interphase microtubule network; 100 nM was used for the destabilizers, eribulin or vinorelbine and 1 µm was used for the stabilizers, paclitaxel or ixabepilone. The results show that TGF-βR1 was extensively localized along the stabilizer-induced microtubule bundles, a phenomenon not observed with destabilizer-treated cells. The downstream consequences were further assessed. The expression of Snail and Slug were evaluated in cells pretreated with MTAs followed by TGF-β stimulation. Eribulin and vinorelbine significantly inhibited the TGF-β-induced expression of Snail and Slug while stabilizers did not alter their expression levels. We hypothesize that eribulin and vinorelbine are inhibiting the non-canonical TGF-β signaling pathway since they had no effect on the localization and expression of Smad2/3, proteins involved in the canonical pathway. Eribulin induced inhibition of TGF-β signaling is consistent with previous studies that show that a 7 day treatment reversed TGF-β mediated EMT in breast cancer cells1. Data from patients shows that eribulin decreases the plasma concentration of TGF-β within 7 days of treatment initiation2. Our data suggest that eribulin rapidly inhibits non-canonical TGF-β signaling indicating that this is a potential mechanism for the eribulin-mediated EMT reversal. This information, together with previously published reports, suggest that eribulin has multiple effects leading to inhibition of TGF-β signaling. These studies begin to shed light into the diverse mechanisms of action of MTAs. 1. Yoshida T, et al., Brit J Cancer 110(6): 1497-505, 2014 2. Ueda S, et al., Brit J Cancer, 2016 This work is funded by Eisai Inc. Citation Format: Kaul R, Risinger AL, Mooberry SL. Eribulin impairs TGF-β type I receptor localization and signaling in BT-549 cells [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P4-04-04.

Nanostructured surfaces of biodegradable silica fibers enhance directed amoeboid cell migration in a microtubule-dependent process

This study reveals significantly enhanced amoeboid cell migration on biodegradable silica fibers in comparison to plain glass surfaces.

PDK1–Akt pathway regulates radial neuronal migration and microtubules in the developing mouse neocortex

Neurons migrate a long radial distance by a process known as locomotion in the developing mammalian neocortex. During locomotion, immature neurons undergo saltatory movement along radial glia fibers. The molecular mechanisms that regulate the speed of locomotion are largely unknown. We now show that the serine/threonine kinase Akt and its activator phosphoinositide-dependent protein kinase 1 (PDK1) regulate the speed of locomotion of mouse neocortical neurons through the cortical plate. Inactivation of the PDK1-Akt pathway impaired the coordinated movement of the nucleus and centrosome, a microtubule-dependent process, during neuronal migration. Moreover, the PDK1-Akt pathway was found to control microtubules, likely by regulating the binding of accessory proteins including the dynactin subunit p150(glued) Consistent with this notion, we found that PDK1 regulates the expression of cytoplasmic dynein intermediate chain and light intermediate chain at a posttranscriptional level in the developing neocortex. Our results thus reveal an essential role for the PDK1-Akt pathway in the regulation of a key step of neuronal migration.

Sequence-dependent but not sequence-specific piRNA adhesion traps mRNAs to the germ plasm.

The conserved Piwi family of proteins and piwi-interacting RNAs (piRNAs) play a central role in genomic stability, which is inextricably tied with germ cell formation, by forming ribonucleoproteins (piRNPs) that silence transposable elements (TEs)1. In Drosophila melanogaster and other animals, primordial germ cell (PGC) specification in the developing embryo is driven by maternal mRNAs and proteins that assemble into specialized mRNPs localized in the germ (pole) plasm at the posterior of the oocyte2,3. Maternal piRNPs, especially those loaded on Aubergine (Aub), a Piwi protein, are transmitted to the germ plasm to initiate transposon silencing in the offspring germline4–7. Transport of mRNAs to the oocyte by midoogenesis is an active, microtubule-dependent process8; mRNAs necessary for PGC formation are enriched in the germ plasm at late oogenesis via a diffusion and entrapment mechanism, whose molecular identity remains unknown8,9. Aub is a central component of germ granule RNPs, which house mRNAs in the germ plasm10–12 and interactions between Aub and Tudor are essential for the formation of germ granules13–16. Here we show that Aub-loaded piRNAs use partial base pairing characteristic of Argonaute RNPs to bind mRNAs randomly, acting as an adhesive trap that captures mRNAs in the germ plasm, in a Tudor-dependent manner. Strikingly, germ plasm mRNAs in Drosophilids are generally longer and more abundant than other mRNAs, suggesting that they provide more target sites for piRNAs to promote their preferential tethering in germ granules. Thus complexes containing Tudor, Aub piRNPs and mRNAs couple piRNA inheritance with germline specification. Our findings reveal an unexpected function for Piwi ribonucleoprotein complexes in mRNA trapping that may be generally relevant to the function of animal germ granules.

Microtubule and Actin Interplay Drive Intracellular c-Src Trafficking

The proto-oncogene c-Src is involved in a variety of signaling processes. Therefore, c-Src spatiotemporal localization is critical for interaction with downstream targets. However, the mechanisms regulating this localization have remained elusive. Previous studies have shown that c-Src trafficking is a microtubule-dependent process that facilitates c-Src turnover in neuronal growth cones. As such, microtubule depolymerization lead to the inhibition of c-Src recycling. Alternatively, c-Src trafficking was also shown to be regulated by RhoB-dependent actin polymerization. Our results show that c-Src vesicles primarily exhibit microtubule-dependent trafficking; however, microtubule depolymerization does not inhibit vesicle movement. Instead, vesicular movement becomes both faster and less directional. This movement was associated with actin polymerization directly at c-Src vesicle membranes. Interestingly, it has been shown previously that c-Src delivery is an actin polymerization-dependent process that relies on small GTPase RhoB at c-Src vesicles. In agreement with this finding, microtubule depolymerization induced significant activation of RhoB, together with actin comet tail formation. These effects occurred downstream of GTP-exchange factor, GEF-H1, which was released from depolymerizing MTs. Accordingly, GEF-H1 activity was necessary for actin comet tail formation at the Src vesicles. Our results indicate that regulation of c-Src trafficking requires both microtubules and actin polymerization, and that GEF-H1 coordinates c-Src trafficking, acting as a molecular switch between these two mechanisms.

Exploring Sialic Acid Receptors‐Related Infection Behavior of Avian Influenza Virus in Human Bronchial Epithelial Cells by Single‐Particle Tracking

Human respiratory tract epithelial cells are the portals of human infection with influenza viruses. However, the infection pathway of individual avian influenza viruses in human respiratory cells remains poorly reported so far. The single-particle tracking technique (SPT) is a powerful tool for studying the transport mechanism of biomolecules in live cells. In this work, we use quantum dots to label avian influenza H9N2 virus and elaborate on the infection mechanism of the virus in human bronchial epithelial (HBE) cells using a three-dimensional SPT technique. We have found that the H9N2 virus can infect HBE cells directly and the virus infection follows an actin filament- and microtubule-dependent process with a three-stage pattern. The transport behaviors show a high degree of consistency between the sialic acid receptors and the influenza virus. Real-time SPT provides dynamic evidence of the sialic acid receptors-related infection behavior of the avian influenza virus in live cells. The study of the influence of sialic acid receptors on virus infection may contribute to a better understanding of the cross-species transmission of the avian influenza virus.

A Novel Role for Progesterone and Progesterone Receptor Membrane Component 1 in Regulating Spindle Microtubule Stability During Rat and Human Ovarian Cell Mitosis

The present studies were designed to assess the roles of progesterone (P4) and Progesterone Receptor Membrane Component 1 (PGRMC1) in regulating mitosis of spontaneously immortalized granulosa cells (SIGCs) and ovarian cancer cells, SKOV-3 cells. Because PGRMC1 has been detected among the proteins of the human mitotic spindle, we theorized that P4 and PGRMC1 could affect mitosis through a microtubule-dependent process. The present study confirms that SIGC growth is slowed by either P4 treatment or transfection of a PGRMC1 antibody. In both cases, slower cell proliferation was accompanied by an increased percentage of mitotic cells, which is consistent with a P4-induced prolongation of the M phase of the cell cycle. In addition, P4 increased the stability of the spindle microtubules, as assessed by the rate of beta-tubulin disassembly in response to cooling. Also, P4 increased spindle microtubule stability of SKOV-3 cells. This effect was mimicked by the depletion of PGRMC1 in these cells. Importantly, P4 did not increase the stability of the microtubules over that observed in PGRMC1-depleted SKOV-3 cells. Immunofluorescent analysis revealed that PGRMC1 is distributed to the spindle apparatus as well as to the centrosomes at metaphase. Further in situ proximity ligation assay revealed that PGRMC1 interacted with beta-tubulin. Taken together, these results suggest that P4 inhibits mitosis of ovarian cells by increasing the stability of the mitotic spindle. Moreover, P4's actions appear to be dependent on PGRMC1's function within the mitotic spindle.

Dynein is a motor for nuclear rotation while vimentin IFs is a “brake”

The positioning of the nucleus is achieved by two interconnected processes, anchoring and migration, both of which are controlled by cytoskeleton structures. Rotation is a special type of nuclear motility in many cell types, but its significance remains unclear. We used a vimentin-null cell line, MFT-16, which shows extensive nuclear rotation to study the phenomenon in detail. By selective disruption of cytoskeletal structures and video-microscopic analysis, nuclear rotation was a microtubule-dependent process that F-actin partially impedes. The dynein-dynactin complex is responsible and inhibiting this motor by expression of a dominant negative mutant of its component P-150 completely stops it. Nuclear rotation is powered by dynein associated with the nuclear envelope along stationary microtubules, centrosomes remaining immobile. We confirmed that vimentin IFs inhibit nuclear rotation, and variant proteins of the mutated wild type gene for vimentin that lacked considerable fragments of the N- and C-terminal domains restored nuclear anchoring. Immunochemical analysis showed that these mutated IFs also bound plectin, arguing for a key role of this cytolinker protein in nuclear anchoring. It is proposed that this versatile machinery guarantees not only rotation and the correct location of a nucleus, but also its orientation in a cell.

Dominant-Negative Myosin Va Impairs Retrograde but Not Anterograde Axonal Transport of Large Dense Core Vesicles

Axonal transport of peptide and hormone-containing large dense core vesicles (LDCVs) is known to be a microtubule-dependent process. Here, we suggest a role for the actin-based motor protein myosin Va specifically in retrograde axonal transport of LDCVs. Using live-cell imaging of transfected hippocampal neurons grown in culture, we measured the speed, transport direction, and the number of LDCVs that were labeled with ectopically expressed neuropeptide Y fused to EGFP. Upon expression of a dominant-negative tail construct of myosin Va, a general reduction of movement in both dendrites and axons was observed. In axons, it was particularly interesting that the retrograde speed of LDCVs was significantly impaired, although anterograde transport remained unchanged. Moreover, particles labeled with the dominant-negative construct often moved in the retrograde direction but rarely in the anterograde direction. We suggest a model where myosin Va acts as an actin-dependent vesicle motor that facilitates retrograde axonal transport.

Assembly of an intact Golgi complex requires phospholipase A 2 (PLA 2) activity, membrane tubules, and dynein-mediated microtubule transport

Previous studies have shown that treatment of mammalian cells with phospholipase A 2 (PLA 2) antagonists cause the normally interconnected Golgi ribbon to break up into large fragments of stacked Golgi cisternae (“mini-stacks”) that remain located in the juxtanuclear region. Using the reversible PLA 2 antagonist, ONO-RS-082 (ONO) and live-cell, time-lapse microscopy to image the Golgi reassembly process, we found that Golgi mini-stacks underwent a burst of membrane tubule formation following washout of ONO: before washout only 4.3 ± 3.8 tubules/cell/10 min were formed, whereas after washout 29.9 ± 11.9 tubules/cell/10 min formed. These membranes tubules formed bridges between physically separate mini-stacks, thus mediating their coalescence into intact Golgi ribbons. Formation of inter-stack tubules and an intact Golgi ribbon was also facilitated by microtubules because treatment with nocodazole significantly inhibited both processes. This microtubule-dependent process was also dependent on dynein because the dynein inhibitor nordihydroguaiaretic acid (NDGA) inhibited reassembly. These studies show that a late stage of Golgi assembly occurs via membrane tubules, whose formation is dependent on PLA 2 activity and microtubules. Considering these results together, we concluded that the maintenance and assembly of normal Golgi architecture is dependent on the PLA 2-mediated, dynamic formation of inter-Golgi membrane tubules.

Actin Dynamics Is Essential for Myosin-Based Transport of Membrane Organelles

Actin filaments that serve as "rails" for the myosin-based transport of membrane organelles [1-4] continuously turn over by concurrent growth and shortening at the opposite ends [5]. Although it is known that dynamics of actin filaments is essential for many of the actin cytoskeleton functions, the role of such dynamics in myosin-mediated organelle transport was never studied before. Here, we addressed the role of turnover of actin filaments in the myosin-based transport of membrane organelles by treating cells with the drugs that suppress actin-filament dynamics and found that such a suppression significantly inhibited organelle transport along the actin filaments without inhibiting their intracellular distribution or the activity of the myosin motors. We conclude that dynamics of actin filaments is essential for myosin-based transport of membrane organelles and suggest a previously unknown role of actin-filament dynamics in providing the "rails" for continuous organelle movement resulting in the increased distances traveled by membrane organelles along the actin filaments.

Dynamics of Somatostatin Type 2A Receptor Cargoes in Living Hippocampal Neurons

Despite the large number of G-protein-coupled receptor (GPCR) types expressed in the CNS, little is known about their dynamics in neuronal cells. Dynamic properties of the somatostatin type 2A receptor were therefore examined in resting conditions and after agonist activation in living hippocampal neurons. Using fluorescence recovery after photobleaching experiments, we found that, in absence of ligand, the sst(2A) receptor is mobile and laterally and rapidly diffuse in neuronal membranes. We then observed by live-cell imaging that, after agonist activation, membrane-associated receptors induce the recruitment of beta-arrestin 1-enhanced green fluorescent protein (EGFP) and beta-arrestin 2-EGFP to the plasma membrane. In addition, beta-arrestin 1-EGFP translocate to the nucleus, suggesting that this protein could serve as a nuclear messenger for the sst(2A) receptor in neurons. Receptors are then recruited to preexisting clathrin coated pits, form clusters that internalize, fuse, and move to a perinuclear compartment that we identified as the trans-Golgi network (TGN), and recycle. Receptor cargoes are transported through a microtubule-dependent process directly from early endosomes/recycling endosomes to the TGN, bypassing the late endosomal compartment. Together, these results provide a comprehensive description of GPCR trafficking in living neurons and provide compelling evidence that GPCR cargoes can recycle through the TGN after endocytosis, a phenomenon that has not been anticipated from studies of non-neuronal cells.

Actin-filament-dependent remodeling of the vacuole in cultured mesophyll protoplasts

The ability of plant cells to dedifferentiate represents an important survival strategy invoked in a range of situations from repair mechanisms following wounding to apomixis. Dedifferentiation requires that somatic cells reprogram and enter the cell division cycle. This in turn necessitates the accurate partitioning of nuclear content and organelles, such as chloroplasts, to daughter cells, thereby ensuring continuity of cellular information systems. The distribution of cytoplasm and its organelle content in mature plant cells is governed by a large, central vacuole, with connections between distant cortical and perinuclear cytoplasmic domains mediated by transvacuolar strands. Here we examined the changes to vacuolar architecture in Arabidopsis thaliana protoplasts expressing a green-fluorescent protein fusion to a delta-tonoplast-intrinsic protein (deltaTIP). We found that vacuolar architecture became increasingly intricate during protoplast culture with the development of numerous transvacuolar strands. The development of an intricate vacuolar architecture was an actin filament- and not microtubule-dependent process, as is the case in interphase plant cells. Furthermore, we show that myosin is required for this increased complexity of vacuolar architecture and the formation of subcortical actin filament arrays. Despite the likelihood that increased vacuolar invagination would allow better redistribution of cytoplasmic organelles, we found that repositioning of chloroplasts from cortical to perinuclear cytoplasm was not dependent on transvacuolar strands. Our findings indicate that the vacuole is a dynamic entity that develops a complex architecture before dedifferentiating plant cells enter cell division.

Differential roles of microtubule assembly and sliding in proplatelet formation by megakaryocytes

Megakaryocytes are terminally differentiated cells that, in their final hours, convert their cytoplasm into long, branched proplatelets, which remodel into blood platelets. Proplatelets elongate at an average rate of 0.85 microm/min in a microtubule-dependent process. Addition of rhodamine-tubulin to permeabilized proplatelets, immunofluorescence microscopy of the microtubule plus-end marker end-binding protein 3 (EB3), and fluorescence time-lapse microscopy of EB3-green fluorescent protein (GFP)-expressing megakaryocytes reveal that microtubules, organized as bipolar arrays, continuously polymerize throughout the proplatelet. In immature megakaryocytes lacking proplatelets, microtubule plus-ends initiate and grow by centrosomal nucleation at rates of 8.9 to 12.3 microm/min. In contrast, plus-end growth rates of microtubules within proplatelets are highly variable (1.5-23.5 microm/min) and are both slower and faster than those seen in immature cells. Despite the continuous assembly of microtubules, proplatelets continue to elongate when net microtubule assembly is arrested. One alternative mechanism for force generation is microtubule sliding. Triton X-100-permeabilized proplatelets containing dynein and its regulatory complex, dynactin, but not kinesin, elongate with the addition of adenosine triphosphate (ATP) at a rate of 0.65 microm/min. Retroviral expression in megakaryocytes of dynamitin (p50), which disrupts dynactin-dynein function, inhibits proplatelet elongation. We conclude that while continuous polymerization of microtubules is necessary to support the enlarging proplatelet mass, the sliding of overlapping microtubules is a vital component of proplatelet elongation.