Geri Kreitzer

Organization of vesicular trafficking in epithelia

Experiments using mammalian epithelial cell lines have elucidated biosynthetic and recycling pathways for apical and basolateral plasma-membrane proteins, and have identified components that guide apical and basolateral proteins along these pathways. These components include apical and basolateral sorting signals, adaptors for basolateral signals, and docking and fusion proteins for vesicular trafficking. Recent live-cell-imaging studies provide a real-time view of sorting processes in epithelial cells, including key roles for actin, microtubules and motors in the organization of post-Golgi trafficking.

Dynein- and Microtubule-Mediated Translocation of Adenovirus Serotype 5 Occurs after Endosomal Lysis

Modified viruses are used as gene transfer vectors because of their ability to transfer genetic material efficiently to the nucleus of a target cell. To better understand intracellular translocation of adenovirus serotype 5 (Ad), fluorophores were covalently conjugated to Ad capsids, and movement of fluorescent Ad within the cytoplasm was observed during the first hour of infection of a human lung epithelial carcinoma cell line (A549). Ad translocation was characterized with respect to its ability to achieve nuclear envelope localization as well as directed movement in the cytoplasm. Whereas Ad achieved efficient nuclear localization 60 min after infection of A549 cells under control conditions, depolymerization of the microtubule cytoskeleton by addition of 25 microM nocodazole reversibly inhibited development of nuclear localization. In contrast, depolymerization of microfilaments by addition of 1 microM cytochalasin D had no effect on nuclear localization. Direct video observation of Ad motility showed that nocodazole, but not cytochalasin D, caused a reversible decrease in rapid linear translocations of Ad in the cytoplasm of A549 cells. Microinjection of function-blocking antibodies against the microtubule-dependent motor protein, cytoplasmic dynein, but not kinesin, blocked nuclear localization of Ad, consistent with net minus end-directed motility indicated by accumulation of Ad at mitotic spindles. Fluorescence ratio imaging revealed a neutral pH in the environment of translocating Ad, leading to a model in which the interaction of Ad with an intact microtubule cytoskeleton and functional cytoplasmic dynein occurs after escape from endosomes and is a necessary prerequisite to nuclear localization of adenovirus serotype 5.

Kinesin and dynamin are required for post-Golgi transport of a plasma-membrane protein

n higher eukaryotes, secretory and plasma-membrane proteins are transported from the endoplasmic reticulum (ER) to a central Golgi complex and subsequently packaged into membrane-bound carriers for delivery to the cell surface. The long-distance transport of post-Golgi organelles to the tips of axons or to developing hyphal extensions in Neurospora crassa shows an absolute requirement for microtubules and microtubule-associated motors. In contrast, microtubule disruption only moderately attenuates Golgi-toplasma-membrane transport in fibroblasts and randomizes surface delivery of select proteins in epithelial cells. The observed preservation of biosynthetic transport after microtubule disruption is probably due to the extensive fragmentation and redistribution of Golgi mini-stacks to regions immediately adjacent to both the ER and the plasma membrane. Here we have designed experiments to test the hypothesis that when the characteristic central localization of the Golgi is preserved, microtubules, kinesin and the GTPase dynamin are essential for post-Golgi trafficking. We ruled out a pharmacological approach to tackling this problem because we found that microtubule antagonists caused dispersal of the Golgi complex before complete microtubule disassembly occurred (see Supplementary Information). Instead, we microinjected functionblocking anti-kinesin antibodies HD and SUK-4 or cDNAs encoding a dominant-negative form of dynamin into cells expressing a green fluorescent protein (GFP)-tagged apical-membrane protein, p75. We show that kinesin and dynamin are required for different stages of post-Golgi transport. During a 2.5-h transport block at 20 °C, newly synthesized p75– GFP translocated from the ER to a juxtanuclear region (Fig. 1a, control, 0 min) and co-localized with Golgi/trans-Golgi network (TGN) markers (Fig. 1b). Within 4 h after shifting to the permissive temperature for transport, 32 °C, 81% of p75–GFP translocated from the Golgi to the plasma membrane (Fig. 1a, control, 240 min). The emptying rate of p75–GFP from the Golgi correlated with its arrival at the cell surface, as determined by immunocytochemical analysis of p75 in injected cells (data not shown) and by pulse– chase, surface-biotinylation assays of p75 or p75–GFP in stable MDCK transfectants (see Supplementary Information). Normal trafficking of p75 was unaffected by the GFP tag: microinjected p75–GFP was selectively delivered to the apical membrane of confluent, polarized MDCK cells (data not shown). I

Three-dimensional analysis of post-Golgi carrier exocytosis in epithelial cells

Targeted delivery of proteins to distinct plasma membrane domains is critical to the development and maintenance of polarity in epithelial cells. We used confocal and time-lapse total internal reflection fluorescence microscopy (TIR-FM) to study changes in localization and exocytic sites of post-Golgi transport intermediates (PGTIs) carrying GFP-tagged apical or basolateral membrane proteins during epithelial polarization. In non-polarized Madin Darby Canine Kidney (MDCK) cells, apical and basolateral PGTIs were present throughout the cytoplasm and were observed to fuse with the basal domain of the plasma membrane. During polarization, apical and basolateral PGTIs were restricted to different regions of the cytoplasm and their fusion with the basal membrane was completely abrogated. Quantitative analysis suggested that basolateral, but not apical, PGTIs fused with the lateral membrane in polarized cells, correlating with the restricted localization of Syntaxins 4 and 3 to lateral and apical membrane domains, respectively. Microtubule disruption induced Syntaxin 3 depolarization and fusion of apical PGTIs with the basal membrane, but affected neither the lateral localization of Syntaxin 4 or Sec6, nor promoted fusion of basolateral PGTIs with the basal membrane.

cdc42 regulates the exit of apical and basolateral proteins from the trans-Golgi network

It is well established that Rho‐GTPases regulate vesicle fusion and fission events at the plasma membrane through their modulatory role on the cortical actin cytoskeleton. In contrast, their effects on intracellular transport processes and actin pools are less clear. It was recently shown that cdc42 associates with the Golgi apparatus in an ARF‐dependent manner, similarly to coat proteins involved in vesicle formation and to several actin‐binding proteins. We report here that mutants of cdc42 inhibited the exit of basolateral proteins from the trans‐Golgi network (TGN), while stimulating the exit of an apical marker, in two different transport assays. This regulation may result from modulation of the actin cytoskeleton, as GTPase‐deficient cdc42 depleted a perinuclear actin pool that rapidly exchanges with exogenous fluorescent actin.

An Annexin 2 Phosphorylation Switch Mediates p11-dependent Translocation of Annexin 2 to the Cell Surface

Annexin 2 is a profibrinolytic co-receptor for plasminogen and tissue plasminogen activator that stimulates activation of the major fibrinolysin, plasmin, at cell surfaces. In human subjects, overexpression of annexin 2 in acute promyelocytic leukemia leads to a bleeding diathesis reflective of excessive cell surface annexin 2-dependent generation of plasmin (Menell, J. S., Cesarman, G. M., Jacovina, A. T., McLaughlin, M. A., Lev, E. A., and Hajjar, K. A. (1999) N. Engl. J. Med. 340, 994–1004). In addition, mice completely deficient in annexin 2 display fibrin accumulation within blood vessels and impaired clearance of injury-induced thrombi (Ling Q., Jacovina, A.T., Deora, A.B., Febbraio, M., Simantov, R., Silverstein, R. L., Hempstead, B. L., Mark, W., and Hajjar, K. A. (2004) J. Clin. Investig. 113, 38–48). Here, we show that endothelial cell annexin 2, a protein that lacks a typical signal peptide, translocates from the cytoplasm to the extracytoplasmic plasma membrane in response to brief temperature stress both in vitro and in vivo in the absence of cell death or cell lysis. This regulated response is independent of new protein or mRNA synthesis and does not require the classical endoplasmic reticulum-Golgi pathway. Temperature stress-induced annexin 2 translocation is dependent on both expression of protein p11 (S100A10) and tyrosine phosphorylation of annexin 2 because annexin 2 release is completely eliminated on depletion of p11, inactivation of tyrosine kinase, or mutation of tyrosine 23. Translocation of annexin 2 to the cell surface dramatically increases tissue plasminogen activator-dependent plasminogen activation potential and may represent a novel stress-induced protein secretion pathway.

Migrating fibroblasts perform polarized, microtubule- dependent exocytosis towards the leading edge

Cell migration might involve biased membrane traffic toward the leading edge to facilitate the building of extracellular matrix, membrane protrusions and adhesion plaques. We tested the hypothesis that secretory vesicles are preferentially delivered toward the leading lamella in wound-edge fibroblasts. Single fusion events of vesicles containing LDLR-GFP were mapped by total internal reflection fluorescence microscopy (TIR-FM). In migrating fibroblasts, exocytic events were polarized towards the leading edge. After disrupting microtubules with nocodazole, exocytosis continued, but fusion sites were clustered around central Golgi elements; there was no peripheral exocytosis. We conclude that microtubules are necessary for the domain-specific fusion of post-Golgi vesicles with the plasma membrane during migration.

LIM Kinase 1 and Cofilin Regulate Actin Filament Population Required for Dynamin-dependent Apical Carrier Fission from the Trans-Golgi Network

The functions of the actin cytoskeleton in post-Golgi trafficking are still poorly understood. Here, we report the role of LIM Kinase 1 (LIMK1) and its substrate cofilin in the trafficking of apical and basolateral proteins in Madin-Darby canine kidney cells. Our data indicate that LIMK1 and cofilin organize a specialized population of actin filaments at the Golgi complex that is selectively required for the emergence of an apical cargo route to the plasma membrane (PM). Quantitative pulse-chase live imaging experiments showed that overexpression of kinase-dead LIMK1 (LIMK1-KD), or of LIMK1 small interfering RNA, or of an activated cofilin mutant (cofilin S3A), selectively slowed down the exit from the trans-Golgi network (TGN) of the apical PM marker p75-green fluorescent protein (GFP) but did not interfere with the apical PM marker glycosyl phosphatidylinositol-YFP or the basolateral PM marker neural cell adhesion molecule-GFP. High-resolution live imaging experiments of carrier formation and release by the TGN and analysis of peri-Golgi actin dynamics using photoactivatable GFP suggest a scenario in which TGN-localized LIMK1-cofilin regulate a population of actin filaments required for dynamin-syndapin-cortactin-dependent generation and/or fission of precursors to p75 transporters.

KIF17 stabilizes microtubules and contributes to epithelial morphogenesis by acting at MT plus ends with EB1 and APC

Cell polarity is determined in part by Kif17-mediated regulation of microtubule dynamics and polymerization rates.

Leukocyte transmigration requires kinesin-mediated microtubule-dependent membrane trafficking from the lateral border recycling compartment

Mamdouh et al. 2008. J. Exp. Med. doi:10.1084/jem.20072328[OpenUrl][1][Abstract/FREE Full Text][2] [1]: {openurl}?query=rft.jtitle%253DJ.%2BExp.%2BMed.%26rft_id%253Dinfo%253Adoi%252F10.1084%252Fjem.20072328%26rft_id%253Dinfo%253Apmid%252F18378793%26rft.genre%253Darticle%26rft_val_fmt%253Dinfo%