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1.  Local cytoskeletal and organelle interactions impact molecular motor-driven early endosomal trafficking 
Current biology : CB  2013;23(13):1173-1180.
In the intracellular environment, motor-driven cargo must navigate a dense cytoskeletal network among abundant organelles.
We investigated the effects of the crowded intracellular environment on early endosomal trafficking. Live-cell imaging of an endosomal cargo (endocytosed epidermal growth factor-conjugated quantum dots) combined with high-resolution tracking was used to analyze the heterogeneous motion of individual endosomes. The motile population of endosomes moved towards the perinuclear region in directed bursts of microtubule-based, dynein-dependent transport interrupted by longer periods of diffusive motion. Actin network density did not affect motile endosomes during directed runs or diffusive interruptions. Simultaneous two-color imaging was used to correlate changes in endosomal movement with potential obstacles to directed runs. Termination of directed runs spatially correlated with microtubule-dense regions, encounters with other endosomes, and interactions with the endoplasmic reticulum. During a subset of run terminations, we also observed merging and splitting of endosomes, deformation of the endoplasmic reticulum, and directional reversals at speeds up to ten-fold greater than characteristic in vitro motor velocities. These observations suggest endosomal membrane tension is high during directed run termination.
Our results indicate that the crowded cellular environment significantly impacts the motor-driven motility of organelles. Rather than simply acting as impediments to movement, interactions of trafficking cargos with intracellular obstacles may facilitate communication between membrane-bound compartments or contribute to the generation of membrane tension necessary for fusion and fission of endosomal membranes or remodeling of the endoplasmic reticulum.
PMCID: PMC3738301  PMID: 23770188
2.  Cell Differentiation through Tissue Elasticity-coupled, Myosin-driven Remodeling 
Current opinion in cell biology  2008;20(6):609-615.
Cells may lack eyes to see and ears to hear, but cells do seem to have a sense of ‘touch’ that allows them to feel their microenvironment. This is achieved in part through contractility-coupled adhesion to physically flexible ‘soft’ tissue. Here we summarize some of the known variations in elasticity of solid tissue and review some of the long-term effects of cells ‘feeling’ this elasticity, focusing on differentiation processes of both committed cell types and stem cells. We then highlight what is known of molecular remodeling in cells under stress on short time scales. Key roles for forces generated by ubiquitous and essential myosin-II motors in feedback remodeling are emphasized throughout.
PMCID: PMC3967913  PMID: 18926907
3.  Imaging the Directed Transport of Single Engineered RNA Transcripts in Real-Time Using Ratiometric Bimolecular Beacons 
PLoS ONE  2014;9(1):e85813.
The relationship between RNA expression and cell function can often be difficult to decipher due to the presence of both temporal and sub-cellular processing of RNA. These intricacies of RNA regulation can often be overlooked when only acquiring global measurements of RNA expression. This has led to development of several tools that allow for the real-time imaging of individual engineered RNA transcripts in living cells. Here, we describe a new technique that utilizes an oligonucleotide-based probe, ratiometric bimolecular beacon (RBMB), to image RNA transcripts that were engineered to contain 96-tandem repeats of the RBMB target sequence in the 3′-untranslated region. Binding of RBMBs to the target RNA resulted in discrete bright fluorescent spots, representing individual transcripts, that could be imaged in real-time. Since RBMBs are a synthetic probe, the use of photostable, bright, and red-shifted fluorophores led to a high signal-to-background. RNA motion was readily characterized by both mean squared displacement and moment scaling spectrum analyses. These analyses revealed clear examples of directed, Brownian, and subdiffusive movements.
PMCID: PMC3893274  PMID: 24454933
4.  Up-regulation of Paxillin and Focal Adhesion Signaling follows Dystroglycan Complex deletions and promotes a Hypertensive State of Differentiation 
European journal of cell biology  2011;90(2-3):249-260.
Anchorage to matrix is mediated for many cells not only by integrin-based focal adhesions but also by a parallel assembly of integral and peripheral membrane proteins known as the Dystroglycan Complex. Deficiencies in either dystrophin (mdx mice) or γ-sarcoglycan (γSG−/− mice) components of the Dystroglycan Complex lead to upregulation of numerous focal adhesion proteins, and the phosphoprotein paxillin proves to be among the most prominent. In mdx muscle, paxillin-Y31 and Y118 are both hyper-phosphorylated as are key sites in focal adhesion kinase (FAK) and the stretch-stimulatable pro-survival MAPK pathway, whereas γSG−/− muscle exhibits more erratic hyper-phosphorylation. In cultured myotubes, cell tension generated by myosin-II appears required for localization of paxillin to adhesions while vinculin appears more stably integrated. Over-expression of wild-type (WT) paxillin has no obvious effect on focal adhesion density or the physical strength of adhesion, but WT and a Y118F mutant promote contractile sarcomere formation whereas a Y31F mutant shows no effect, implicating Y31 in striation. Self-peeling of cells as well as Atomic Force Microscopy (AFM) probing of cells with or without myosin II inhibition indicate an increase in cell tension within paxillin-overexpressing cells. However, prednisolone, a first-line glucocorticoid for muscular dystrophies, decreases cell tension without affecting paxillin at adhesions, suggesting a non-linear relationship between paxillin and cell tension. Hypertension that results from upregulation of integrin adhesions is thus a natural and treatable outcome of dystroglycan complex down-regulation.
PMCID: PMC2970638  PMID: 20663583
adhesion; contractility; differentiation; paxillin; muscular dystrophy; hypertensive; glucocorticoid
5.  Huntingtin coordinates the dynein-mediated dynamic positioning of endosomes and lysosomes 
Molecular Biology of the Cell  2011;22(4):478-492.
We investigated the role of the membrane-associated scaffolding protein huntingtin (Htt) in the dynein-mediated transport of early, recycling, and late endosomes and lysosomes. Our observations support a model of Htt as a facilitator of dynein-mediated trafficking that can regulate the cytoskeletal association of dynamic organelles.
Huntingtin (Htt) is a membrane-associated scaffolding protein that interacts with microtubule motors as well as actin-associated adaptor molecules. We examined a role for Htt in the dynein-mediated intracellular trafficking of endosomes and lysosomes. In HeLa cells depleted of either Htt or dynein, early, recycling, and late endosomes (LE)/lysosomes all become dispersed. Despite altered organelle localization, kinetic assays indicate only minor defects in intracellular trafficking. Expression of full-length Htt is required to restore organelle localization in Htt-depleted cells, supporting a role for Htt as a scaffold that promotes functional interactions along its length. In dynein-depleted cells, LE/lysosomes accumulate in tight patches near the cortex, apparently enmeshed by cortactin-positive actin filaments; Latrunculin B-treatment disperses these patches. Peripheral LE/lysosomes in dynein-depleted cells no longer colocalize with microtubules. Htt may be required for this off-loading, as the loss of microtubule association is not seen in Htt-depleted cells or in cells depleted of both dynein and Htt. Inhibition of kinesin-1 relocalizes peripheral LE/lysosomes induced by Htt depletion but not by dynein depletion, consistent with their detachment from microtubules upon dynein knockdown. Together, these data support a model of Htt as a facilitator of dynein-mediated trafficking that may regulate the cytoskeletal association of dynamic organelles.
PMCID: PMC3038646  PMID: 21169558
6.  Novel ANKH Mutation in a Patient with Sporadic Craniometaphyseal Dysplasia 
Craniometaphyseal dysplasia is caused by mutations in ANKH (ankylosis, progressive homolog (mouse)) in the majority of cases, and all of the reported mutations are single amino acid changes. Genomic DNA from an affected patient, his biological parents and a sibling was amplified and ANKH was sequenced. The affected patient had a complex heterozygous mutation in exon 7 (c.936T>C, c.938C>G, c.942_953delTGGTTGACGGAA), predicting p.Try290Gln and p.Trp292_Glu295del. We studied the effect of the predicted mutation on the subcellular distribution of ANKH protein. Immunofluorescent labeling of COS-7 cells transduced with normal or mutant Ank (murine progressive ankylosis), showed that normal Ank localized to both the plasma membrane and cytoplasm, while mutant Ank was detected only in the cytoplasmic compartment. We propose that this craniometaphyseal dysplasia mutation causes a loss of ANKH protein expression and activity in the plasma membrane due to aberrant intracellular protein trafficking.
PMCID: PMC2944898  PMID: 20186813
Craniometaphyseal dysplasia; ANKH; deletion mutation; intracellular protein trafficking
7.  Different Localizations and Cellular Behaviors of Leiomodin and Tropomodulin in Mature Cardiomyocyte Sarcomeres 
Molecular Biology of the Cell  2010;21(19):3352-3361.
Lmod is a muscle-specific actin nucleator that displays structural similarity to the filament pointed-end–capping protein, Tmod. The mechanisms of localizations of Lmod and Tmod in muscle sarcomeres are strikingly different. Lmod contributes to the organization of mature myofibrils through a mechanism that requires interaction with tropomyosin.
Leiomodin (Lmod) is a muscle-specific F-actin–nucleating protein that is related to the F-actin pointed-end–capping protein tropomodulin (Tmod). However, Lmod contains a unique ∼150-residue C-terminal extension that is required for its strong nucleating activity. Overexpression or depletion of Lmod compromises sarcomere organization, but the mechanism by which Lmod contributes to myofibril assembly is not well understood. We show that Tmod and Lmod localize through fundamentally different mechanisms to the pointed ends of two distinct subsets of actin filaments in myofibrils. Tmod localizes to two narrow bands immediately adjacent to M-lines, whereas Lmod displays dynamic localization to two broader bands, which are generally more separated from M-lines. Lmod's localization and F-actin nucleation activity are enhanced by interaction with tropomyosin. Unlike Tmod, the myofibril localization of Lmod depends on sustained muscle contraction and actin polymerization. We further show that Lmod expression correlates with the maturation of myofibrils in cultured cardiomyocytes and that it associates with sarcomeres only in differentiated myofibrils. Collectively, the data suggest that Lmod contributes to the final organization and maintenance of sarcomere architecture by promoting tropomyosin-dependent actin filament nucleation.
PMCID: PMC2947471  PMID: 20685966
8.  Flexible Filaments for in vivo Imaging and Delivery 
Molecular pharmaceutics  2009;6(5):1343-1352.
Shape effects of synthetic carriers are largely unexplored in vivo, although recent findings suggest that flexible filaments can persist in the circulation even if microns in length. Here, to better assess biodistribution, a near infrared fluorophore (NIRF) was incorporated into such block copolymer ‘filomicelles’, and both in vivo and ex vivo imaging show that the majority of these worm-like micelles remain in the circulation for at least a day after intravenous injection. NIRF imaging further suggests that filomicelles convect into a tumor and some fragments can penetrate into the tumor stroma. To assess a functional effect, the hydrophobic drug paclitaxel (tax) was loaded into both filomicelles and sonication-generated spherical micelles of the same copolymer. Intravenous injection of tax-loaded filomicelles nearly doubles the maximum tolerated dose of tax in normal mice compared to tax-loaded spherical micelles. In tumor-bearing mice, the higher dose of tax produces greater and more sustained tumor shrinkage and tumor cell apoptosis. These results thus begin to address mechanisms for how non-spherical carriers deliver both imaging agents and anti-cancer therapeutics to solid tumors.
PMCID: PMC2757530  PMID: 19249859
9.  Membrane association and functional regulation of Sec3 by phospholipids and Cdc42 
The Journal of Cell Biology  2008;180(1):145-158.
The exocyst is an octameric protein complex implicated in tethering post-Golgi secretory vesicles at the plasma membrane in preparation for fusion. However, it is not clear how the exocyst is targeted to and physically associates with specific domains of the plasma membrane and how its functions are regulated at those regions. We demonstrate that the N terminus of the exocyst component Sec3 directly interacts with phosphatidylinositol 4,5-bisphosphate. In addition, we have identified key residues in Sec3 that are critical for its binding to the guanosine triphosphate–bound form of Cdc42. Genetic analyses indicate that the dual interactions of Sec3 with phospholipids and Cdc42 control its function in yeast cells. Disrupting these interactions not only blocks exocytosis and affects exocyst polarization but also leads to defects in cell morphogenesis. We propose that the interactions of Sec3 with phospholipids and Cdc42 play important roles in exocytosis and polarized cell growth.
PMCID: PMC2213614  PMID: 18195105
10.  Cyclical Regulation of the Exocyst and Cell Polarity Determinants for Polarized Cell Growth 
Molecular Biology of the Cell  2005;16(3):1500-1512.
Polarized exocytosis is important for morphogenesis and cell growth. The exocyst is a multiprotein complex implicated in tethering secretory vesicles at specific sites of the plasma membrane for exocytosis. In the budding yeast, the exocyst is localized to sites of bud emergence or the tips of small daughter cells, where it mediates secretion and cell surface expansion. To understand how exocytosis is spatially controlled, we systematically analyzed the localization of Sec15p, a member of the exocyst complex and downstream effector of the rab protein Sec4p, in various mutants. We found that the polarized localization of Sec15p relies on functional upstream membrane traffic, activated rab protein Sec4p, and its guanine exchange factor Sec2p. The initial targeting of both Sec4p and Sec15p to the bud tip depends on polarized actin cable. However, different recycling mechanisms for rab and Sec15p may account for the different kinetics of polarization for these two proteins. We also found that Sec3p and Sec15p, though both members of the exocyst complex, rely on distinctive targeting mechanisms for their localization. The assembly of the exocyst may integrate various cellular signals to ensure that exocytosis is tightly controlled. Key regulators of cell polarity such as Cdc42p are important for the recruitment of the exocyst to the budding site. Conversely, we found that the proper localization of these cell polarity regulators themselves also requires a functional exocytosis pathway. We further report that Bem1p, a protein essential for the recruitment of signaling molecules for the establishment of cell polarity, interacts with the exocyst complex. We propose that a cyclical regulatory network contributes to the establishment and maintenance of polarized cell growth in yeast.
PMCID: PMC551511  PMID: 15647373

Results 1-10 (10)