Biomedical subjects

Derek Toomre

Publications and source records attributed to Derek Toomre.

13 recordsLinked to original sources

PubMed2006

Two synaptojanin 1 isoforms are recruited to clathrin-coated pits at different stages.

Phosphoinositides are thought to play an important role in clathrin-coated pit (CCP) dynamics. Biochemical and structural studies have shown a direct interaction of phosphatidylinositol (4,5)-bisphosphate [PI(4,5)P2] with endocytic clathrin adaptors, whereas functional studies using cell-free systems or intact cells have demonstrated the importance of PI(4,5)P2 synthesis and dephosphorylation in clathrin coating and uncoating, respectively. Furthermore, genetic manipulations of kinases and phosphatases involved in PI(4,5)P2 metabolism result in major defects in synaptic vesicle recycling and other forms of clathrin-dependent endocytosis. However, live imaging studies of these enzymes at CCPs have not been conducted. We have used multicolor total internal reflection fluorescence microscopy (TIRFM) to visualize the spatial-temporal recruitment of synaptojanin 1 (SJ1), a polyphosphoinositide phosphatase, and its binding partner endophilin to CCPs. Strikingly, we observed differential temporal recruitment of the two major SJ1 splice variants to CCPs. The 145-kDa isoform, the predominant isoform expressed in the brain, was rapidly recruited as a "burst," together with endophilin, at a late stage of CCP formation. In contrast, the nonneuronal ubiquitously expressed 170-kDa isoform of SJ1 was present at all stages of CCP formation. These results raise the possibility that dynamic phosphoinositide metabolism may occur throughout the lifetime of a CCP.

Acyltransferases↗

PubMed2006

Vectorial insertion of apical and basolateral membrane proteins in polarized epithelial cells revealed by quantitative 3D live cell imaging.

Although epithelial cells are known to exhibit a polarized distribution of membrane components, the pathways responsible for delivering membrane proteins to their appropriate domains remain unclear. Using an optimized approach to three-dimensional live cell imaging, we have visualized the transport of newly synthesized apical and basolateral membrane proteins in fully polarized filter-grown Madin-Darby canine kidney cells. We performed a detailed quantitative kinetic analysis of trans-Golgi network (TGN) exit, passage through transport intermediates, and arrival at the plasma membrane using cyan/yellow fluorescent protein-tagged glycosylphosphatidylinositol-anchored protein and vesicular stomatitis virus glycoprotein as apical and basolateral reporters, respectively. For both pathways, exit from the TGN was rate limiting. Furthermore, apical and basolateral proteins were targeted directly to their respective membranes, resolving current confusion as to whether sorting occurs on the secretory pathway or only after endocytosis. However, a transcytotic protein did reach the apical surface after a prior appearance basolaterally. Finally, newly synthesized proteins appeared to be delivered to the entire lateral or apical surface, suggesting-contrary to expectations-that there is not a restricted site for vesicle docking or fusion adjacent to the junctional complex.

Animals↗

PubMed2006

Lymphocyte transcellular migration occurs through recruitment of endothelial ICAM-1 to caveola- and F-actin-rich domains.

During inflammation, leukocytes bind to the adhesion receptors ICAM-1 and VCAM-1 on the endothelial surface before undergoing transendothelial migration, also called diapedesis. ICAM-1 is also involved in transendothelial migration, independently of its role in adhesion, but the molecular basis of this function is poorly understood. Here we demonstrate that, following clustering, apical ICAM-1 translocated to caveolin-rich membrane domains close to the ends of actin stress fibres. In these F-actin-rich areas, ICAM-1 was internalized and transcytosed to the basal plasma membrane through caveolae. Human T-lymphocytes extended pseudopodia into endothelial cells in caveolin- and F-actin-enriched areas, induced local translocation of ICAM-1 and caveolin-1 to the endothelial basal membrane and transmigrated through transcellular passages formed by a ring of F-actin and caveolae. Reduction of caveolin-1 levels using RNA interference (RNAi) specifically decreased lymphocyte transcellular transmigration. We propose that the translocation of ICAM-1 to caveola- and F-actin-rich domains links the sequential steps of lymphocyte adhesion and transendothelial migration and facilitates lymphocyte migration through endothelial cells from capillaries into surrounding tissue.

Actins↗

PubMed2006

Overlapping events with application to image sequences.

Counting spatially and temporally overlapping events in image sequences and estimating their shape-size and duration features are important issues in some applications. We propose a stochastic model, a particular case of the nonisotropic 3D Boolean model, for performing this analysis: the temporal Boolean model. Some probabilistic properties are derived and a methodology for parameter estimation from time-lapse image sequences is proposed using an explicit treatment of the temporal dimension. We estimate the mean number of germs per unit area and time, the mean grain size and the duration distribution. A wide simulation study in order to assess the proposed estimators showed promising results. The model was applied on biological image sequences of in-vivo cells in order to estimate new parameters such as the mean number and duration distribution of endocytic events. Our results show that the proposed temporal Boolean model is effective for obtaining information about dynamic processes which exhibit short-lived, but spatially and temporally overlapping events.

Algorithms↗

PubMed2005

Spying on IgE receptor signaling: simply complex, or not?

Plasma membrane organization and the potential role, or not, of lipid raft microdomains in signal transduction is a controversial topic. Cross-correlation fluorescent correlation spectroscopy (CC-FCS) shows promise as a new approach to rapidly probe protein-protein interactions in living cells during signal transduction. CC-FCS data from studies of IgE receptor signaling challenge models of large stable lipid raft signaling domains and reveal a new complexity in the dynamic (re)organization of signaling complexes.

Animals↗

PubMed2005

Phosphatidylinositol phosphate kinase type I gamma regulates dynamics of large dense-core vesicle fusion.

Phosphatidylinositol-4,5-bisphosphate was proposed to be an important regulator of large dense-core vesicle exocytosis from neuroendocrine tissues. Here, we have examined the kinetics of secretion in chromaffin cells from mice lacking phosphatidylinositol phosphate kinase type I gamma, the major neuronal phosphatidylinositol-4-phosphate 5-kinase. Absence of this enzyme caused a reduction of the readily releasable vesicle pool and its refilling rate, with a small increase in morphologically docked vesicles, indicating a defect in vesicle priming. Furthermore, amperometry revealed a delay in fusion pore expansion. These results provide direct genetic evidence for a key role of phosphatidylinositol-4,5-bisphosphate synthesis in the regulation of large dense-core vesicle fusion dynamics.

Animals↗

PubMed2005

The Abl/Arg substrate ArgBP2/nArgBP2 coordinates the function of multiple regulatory mechanisms converging on the actin cytoskeleton.

ArgBP2, and its brain-specific splice variant, nArgBP2, are interactors and substrates of Abl/Arg tyrosine kinases and of the ubiquitin ligase Cbl. They are members of a family of adaptor proteins that colocalize with actin on stress fibers and at cell-adhesion sites, including neuronal synapses. We show here that their NH2-terminal region, which contains a sorbin homology domain domain, interacts with spectrin, and we identify binding proteins for their COOH-terminal SH3 domains. All these binding partners participate in the regulation of the actin cytoskeleton. These include dynamin, synaptojanin, and WAVE isoforms, as well as WAVE regulatory proteins. At least two of the ArgBP2/nArgBP2 binding partners, synaptojanin 2B and WAVE2, undergo ubiquitination and Abl-dependent tyrosine phosphorylation. ArgBP2/nArgBP2 knockdown in astrocytes produces a redistribution of focal adhesion proteins and an increase in peripheral actin ruffles, whereas nArgBP2 overexpression produces a collapse of the actin cytoskeleton. Thus, ArgBP2/nArgBP2 is a scaffold protein that control the balance between adhesion and motility by coordinating the function of multiple signaling pathways converging on the actin cytoskeleton.

Actins↗

PubMed2005

Segmentation and 3D reconstruction of microtubules in total internal reflection fluorescence microscopy (TIRFM).

The interaction of the microtubules with the cell cortex plays numerous critical roles in a cell. For instance, it directs vesicle delivery, and modulates membrane adhesions pivotal for cell movement as well as mitosis. Abnormal function of the microtubules is involved in cancer. An effective method to observe microtubule function adjacent to the cortex is TIRFM. To date most analysis of TIRFM images has been done by visual inspection and manual tracing. In this work we have developed a method to automatically process TIRFM images of microtubules so as to enable high throughput quantitative studies. The microtubules are extracted in terms of consecutive segments. The segments are described via Hamilton-Jacobi equations. Subsequently, the algorithm performs a limited reconstruction of the microtubules in 3D. Last, we evaluate our method with phantom as well as real TIRFM images of living cells.

Algorithms↗

PubMed2004

Golgi duplication in Trypanosoma brucei.

Duplication of the single Golgi apparatus in the protozoan parasite Trypanosoma brucei has been followed by tagging a putative Golgi enzyme and a matrix protein with variants of GFP. Video microscopy shows that the new Golgi appears de novo, near to the old Golgi, about two hours into the cell cycle and grows over a two-hour period until it is the same size as the old Golgi. Duplication of the endoplasmic reticulum (ER) export site follows exactly the same time course. Photobleaching experiments show that the new Golgi is not the exclusive product of the new ER export site. Rather, it is supplied, at least in part, by material directly from the old Golgi. Pharmacological experiments show that the site of the new Golgi and ER export is determined by the location of the new basal body.

Animals↗

PubMed2002

Dendritic cell maturation triggers retrograde MHC class II transport from lysosomes to the plasma membrane.

Central to the initiation of immune responses is recognition of peptide antigen by T lymphocytes. The cell biology of dendritic cells makes them ideally suited for the essential process of antigen presentation. Their life cycle includes several stages characterized by distinct functions and mechanisms of regulation. Immature dendritic cells synthesize large amounts of major histocompatibility complex class II molecules (MHC II), but the alpha beta-dimers are targeted to late endosomes and lysosomes (often referred to as MHC class II compartments) where they reside unproductively with internalized antigens. After exposure to microbial products or inflammatory mediators, endocytosis is downregulated, the expression of co-stimulatory molecules is enhanced, and newly formed immunogenic MHC II-peptide complexes are transported to the cell surface. That these MHC II molecules reach the surface is surprising, as the lysosomes comprise the terminal degradative compartment of the endocytic pathway from which exogenous components generally cannot be recovered intact. Here we have visualized this pathway in live dendritic cells by video microscopy, using cells expressing MHC II tagged with green fluorescent protein (GFP). We show that on stimulation, dendritic cells generate tubules from lysosomal compartments that go on to fuse directly with the plasma membrane.

Animals↗

PubMed2002

Golgi biogenesis in Toxoplasma gondii.

Two models have been put forward to explain the growth of new Golgi during the cell cycle. The first suggests that a new Golgi grows out of the endoplasmic reticulum by de novo synthesis. The second suggests that a pre-existing Golgi is needed for the growth of a new one, that is, the Golgi is an autonomously replicating organelle. To resolve this issue, we have exploited the simplicity of the apicomplexan parasite Toxoplasma gondii, which has only a single Golgi stack. Here we show, by using video fluorescence microscopy and three-dimensional reconstructions of serial thin sections, that the Golgi grows by a process of lateral extension followed by medial fission. Further fission leads to the inheritance by each daughter of a pair of Golgi structures, which then coalesce to re-form a single Golgi. Our results indicate that new Golgi grow by autonomous duplication and raise the possibility that the Golgi is a paired structure that is analogous to centrioles.

Animals↗

PubMed2002

Selective delivery of secretory cargo in Golgi-derived carriers of nonepithelial cells.

In epithelial cells, soluble cargo proteins destined for basolateral or apical secretion are packaged into distinct trans-Golgi network-derived transport carriers. Similar carriers, termed basolateral- and apical-like, have been observed in nonepithelial cells using ectopically expressed membrane marker proteins. Whether these cells are capable of selectively packaging secretory proteins into distinct carriers is still an open question. Here, we have addressed this issue by analyzing the packaging and transport of secretory human chromogranin B fusion proteins using a green fluorescent protein-based high-resolution, dual-color imaging technique. We were able to show that these secretory markers were selectively packaged at the Golgi into tubular/vesicular-like transport carriers containing basolateral membrane markers, resulting in extensive cotransport. In contrast, deletion mutants of the human chromogranin B fusion proteins lacking an N-terminal loop structure were efficiently transported in both basolateral- and apical-like carriers, the latter displaying a spherical morphology. Similarly, in polarized epithelial cells, the human chromogranin B fusion protein was secreted basolaterally and the loop-deleted analogue into both the basolateral and apical medium. These findings suggest that nonepithelial cells, like their epithelial counterparts, possess a sorting machinery capable of selective packaging of secretory cargo into distinct types of carriers.

Animals↗

PubMedDate not supplied

Spatio-temporal analysis of constitutive exocytosis in epithelial cells.

Exocytosis is an essential cellular trafficking process integral to the proper distribution and function of a plethora of molecules, including transporters, receptors, and enzymes. Moreover, incorrect protein targeting can lead to pathological conditions. Recently, the application of evanescent wave microscopy has allowed us to image the final steps of exocytosis. However, spatio-temporal analysis of fusion of constitutive vesicular traffic with the plasma membrane has not been systematically performed. Also, the spatial sites and times of vesicle fusion have not yet been analyzed together. In addition, more formal tests are required in testing biological hypotheses, rather than visual inspection combined with statistical descriptives. Ripley K-functions are used to examine the joint and marginal behavior of locations and fusion times. Semiautomatic detection and mapping of constitutive fusion sites reveals spatial and temporal clustering, but no dependency between the locations and times of fusion events. Our novel approach could be translated to other studies of membrane trafficking in health and diseases such as diabetes.

Cell Line↗