Calcium-dependent protein kinases regulate polarized tip growth in pollen tubes

Calcium signals are critical for the regulation of polarized growth in many eukaryotic cells, including pollen tubes and neurons. In plants, the regulatory pathways that code and decode Ca²⁺ signals are poorly understood. In Arabidopsis thaliana, genetic evidence presented here indicates that pollen tube tip growth involves the redundant activity of two Ca²⁺-dependent protein kinases (CPKs), isoforms CPK17 and -34. Both isoforms appear to target to the plasma membrane, as shown by imaging of CPK17–yellow fluorescent protein (YFP) and CPK34–YFP in growing pollen tubes. Segregation analyses from two independent sets of T-DNA insertion mutants indicate that a double disruption of CPK17 and -34 results in an approximately 350-fold reduction in pollen transmission efficiency. The near sterile phenotype of homozygous double mutants could be rescued through pollen expression of a CPK34–YFP fusion. In contrast, a transgene rescue was blocked by mutations engineered to disrupt the Ca²⁺-activation mechanism of CPK34 (CPK34–YFP–E465A,E500A), providing in vivo evidence linking Ca²⁺ activation to a biological function of a CPK. While double mutant pollen tubes displayed normal morphology, relative growth rates for the most rapidly growing tubes were reduced by more than three-fold compared with wild type. In addition, while most mutant tubes appeared to grow far enough to reach ovules, the vast majority (>90%) still failed to locate and fertilize ovules. Together, these results provide genetic evidence that CPKs are essential to pollen fitness, and support a mechanistic model in which CPK17 and -34 transduce Ca²⁺ signals to increase the rate of pollen tube tip growth and facilitate a response to tropism cues.     

Inhibition of actin polymerisation by low concentration Latrunculin B affects endocytosis and alters exocytosis in shank and tip of tobacco pollen tubes

Pollen tube growth depends on the integrity of the actin cytoskeleton that regulates cytoplasmic streaming and secretion. To clarify whether actin also plays a role in pollen tube endocytosis, Latrunculin B (LatB) was employed in internalisation experiments with tobacco pollen tubes, using the lipophilic dye FM4‐64 and charged nanogold. Time‐lapse analysis and dissection of endocytosis allowed us to identify internalisation pathways with different sensitivity to LatB. Co‐localisation experiments and ultrastructural observations using positively charged nanogold revealed that LatB significantly inhibited endocytosis in the pollen tube shank, affecting internalisation of the plasma membrane (PM) recycled for secretion, as well as that conveyed to vacuoles. In contrast, endocytosis of negatively charged nanogold in the tip, which is also conveyed to vacuoles, was not influenced. Experiments of fluorescence recovery after photobleaching (FRAP) of the apical and subapical PM revealed domains with different rates of fluorescence recovery and showed that these differences depend on the actin cytoskeleton integrity. These results show the presence of distinct degradation pathways by demonstrating that actin‐dependent and actin‐indepedent endocytosis both operate in pollen tubes, internalising tracts of PM to be recycled and broken down. Intriguingly, although most studies concentrate on exocytosis and distension in the apex, the present paper shows that uncharacterised, actin‐dependent secretory activity occurs in the shank of pollen tubes.     

Vesicle trafficking dynamics and visualization of zones of exocytosis and endocytosis in tobacco pollen tubes

Pollen tubes are one of the fastest growing eukaryotic cells.Rapid anisotropic growth is supported by highly active exocytosisand endocytosis at the plasma membrane, but the subcellularlocalization of these sites is unknown. To understand molecularprocesses involved in pollen tube growth, it is crucial to identifythe sites of vesicle localization and trafficking. This reportpresents novel strategies to identify exocytic and endocyticvesicles and to visualize vesicle trafficking dynamics, usingpulse-chase labelling with styryl FM dyes and refraction-freehigh-resolution time-lapse differential interference contrastmicroscopy. These experiments reveal that the apex is the siteof endocytosis and membrane retrieval, while exocytosis occursin the zone adjacent to the apical dome. Larger vesicles areinternalized along the distal pollen tube. Discretely sizedvesicles that differentially incorporate FM dyes accumulatein the apical, subapical, and distal regions. Previous workestablished that pollen tube growth is strongly correlated withhydrodynamic flux and cell volume status. In this report, itis shown that hydrodynamic flux can selectively increase exocytosisor endocytosis. Hypotonic treatment and cell swelling stimulatedexocytosis and attenuated endocytosis, while hypertonic treatmentand cell shrinking stimulated endocytosis and inhibited exocytosis.Manipulation of pollen tube apical volume and membrane remodellingenabled fine-mapping of plasma membrane dynamics and definedthe boundary of the growth zone, which results from the orchestratedaction of endocytosis at the apex and along the distal tubeand exocytosis in the subapical region. This report providescrucial spatial and temporal details of vesicle traffickingand anisotropic growth. Key words: Endocytosis; exocytosis, hydrodynamics, lipophilic FM dyes, pollen tube growth, vesicle trafficking Received 14 September 2007; Revised 23 November 2007 Accepted 7 January 2008     

Specific Grb2-mediated interactions regulate clathrin-dependent endocytosis of the cMet-tyrosine kinase

Lysosomal degradation of the receptor-tyrosine kinase cMet requires receptor ubiquitination by the E3 ubiquitin ligase Cbl followed by clathrin-dependent internalization. A role for Cbl as an adaptor for cMet internalization has been previously reported. However, the requirement for Cbl ubiquitin ligase activity in this process and its mode of recruitment to cMet has yet to be determined. Cbl can directly bind cMet at phosphotyrosine 1003 or indirectly via Grb2 to phosphotyrosine 1356 in the multisubstrate binding domain of cMet. The direct binding of Cbl with cMet is critical for receptor degradation and not receptor internalization. Here we show a strict requirement for Grb2 and the ubiquitin ligase activity of Cbl for cMet endocytosis. Receptor internalization was impaired by small interfering RNA depletion of Grb2, overexpression of dominant negative Grb2 mutants, and point mutations in the cMet multisubstrate docking site that inhibits the direct association of Grb2 with cMet. The requirement for Grb2 was specific and did not involve the multiadaptor Gab1. cMet internalization was impaired in cells expressing an ubiquitin ligase-deficient Cbl mutant or conjugation-deficient ubiquitin but was unaffected in cells expressing a Cbl mutant that is unable to bind cMet directly. Expression of a Cbl-Grb2 chimera rescued impaired cMet endocytosis in cells depleted of endogenous Grb2. These results indicate that the ubiquitin ligase activity of Cbl is critical for clathrin-dependent cMet internalization and suggest a role for Grb2 as an intermediary linking Cbl ubiquitin ligase activity to this process.     

Quantitative analysis of the distribution of organelles in tobacco pollen tubes: implications for exocytosis and endocytosis

Summary The present study provides the first quantitative analysis on the distribution of organelles in pollen tubes ofNicotiana tabacum L. Organelles were studied on living pollen tubes by means of fluorescence confocal laser scanning microscopy and on cryo-fixed, freeze-substituted and serially sectioned material by electron microscopy. In the tip a 300 nm to 400 nm thick wall was secreted that proximately gradually separated into a wall with an opaque inner side and a more translucent, layered outer side. Tubular endoplasmic reticulum was particularly abundant in the tip of the tube, surrounding the region where secretory vesicles (SV) accumulated. Mitochondria were randomly distributed throughout the cytoplasm, no accumulations were present. Dictyosomes, however, showed an increased abundance at 25–30 m behind the tip. The accumulation of coated pits (CP) in a zone 6–15 m behind the tip identifies this zone as the major site of endocytosis: 50% of all CP occur in this zone. Quantification of exo- and endocytosis showed that only part of the membrane material of the SV can be retrieved after exocytosis. The typical zonation in endocytotic activity may serve to maintain a difference in membrane protein composition between the tip and the tube.     

Cross-talk between clathrin-dependent post-Golgi trafficking and clathrin-mediated endocytosis in Arabidopsis root cells

Coupling of post-Golgi and endocytic membrane transport ensures that the flow of materials to/from the plasma membrane (PM) is properly balanced. The mechanisms underlying the coordinated trafficking of PM proteins in plants, however, are not well understood. In plant cells, clathrin and its adaptor protein complexes, AP-2 and the TPLATE complex (TPC) at the PM, and AP-1 at the trans-Golgi network/early endosome (TGN/EE), function in clathrin-mediated endocytosis (CME) and post-Golgi trafficking. Here, we utilized mutants with defects in clathrin-dependent post-Golgi trafficking and CME, in combination with other cytological and pharmacological approaches, to further investigate the machinery behind the coordination of protein delivery and recycling to/from the TGN/EE and PM in Arabidopsis (Arabidopsis thaliana) root cells. In mutants with defective AP-2-/TPC-dependent CME, we determined that clathrin and AP-1 recruitment to the TGN/EE as well as exocytosis are significantly impaired. Likewise, defects in AP-1-dependent post-Golgi trafficking and pharmacological inhibition of exocytosis resulted in the reduced association of clathrin and AP-2/TPC subunits with the PM and a reduction in the internalization of cargoes via CME. Together, these results suggest that post-Golgi trafficking and CME are coupled via modulation of clathrin and adaptor protein complex recruitment to the TGN/EE and PM.     

Localized endocytosis in tobacco pollen tubes: visualisation and dynamics of membrane retrieval by a fluorescent phospholipid

Two modes of endocytosis are known to occur in eucaryotic cells: fluid phase and receptor-mediated endocytosis. Fluid-phase endocytosis in plant cells resembles the retrieval of excess plasma membrane material previously incorporated by exocytosis. Pollen tubes need to carry out strong membrane retrieval due to their fast polar tip growth. Plasma membrane labelling of pollen tubes, grown in suspension, was achieved by the incorporation of a fluorescently modified phospholipid, 1,2-bis-(4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-undecanoyl)-sn-glycero-3-phosphocholine (20 μM) and measured with a confocal laser-scanning microscope. Time course experiments revealed a highly localised and relatively fast plasma membrane retrieval below the tip within the first 5 min after phospholipid application. The retrieved fluorescent plasma membrane was quickly re-integrated into parts of the endomembrane pool and then redistributed to the pollen tube base and very tip of the apex, with the exception of the cortical endoplasmic reticulum (ER) and the mitochondria even after 1-h incubation period. Low temperature (10°C) and the actin filament depolymerizing cytochalasin D (2 μM) completely abolished plasma membrane retrieval, whereas the microtubule destabilizing herbicide oryzalin (1 μM) had no effect. Our results provide strong support for a highly localised endocytotic pathway in tobacco pollen tubes. Passive uptake of bis-Bodipy FL C₁₁-phosphocholine by mere penetration can be excluded. It is a valuable alternative to the styryl dyes often used in endocytotic studies, and may also be used to follow lipid turnover because membrane flow of labelled membranes occurs apparently not in a default manner as ascertained by its fast distribution. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Exocytosis in non-plasmolyzed and plasmolyzed tobacco pollen tubes

Exocytosis occurring during deposition of secondary wall material was studied by freeze-fracturing ultrarapidly frozen non-plasmolyzed and plasmolyzed tobacco pollen tubes. The secondary wall of tobacco pollen tubes shows a random orientation of microfibrils. This was observed directly on fractures through the tube wall and indirectly as imprints of microfibrils on fracture faces of the plasma membrane of non-plasmolyzed tubes. About half of the plasmatic fracture faces from non-plasmolyzed and plasmolyzed pollen tubes carried hexagonal arrays of intramembraneous particles in between randomly distributed particles. Deposition of secondary wall material was often accompanied by an undulated plasma membrane and the presence of membrane-bound vesicles in invaginations of the plasma membrane, between the plasma membrane and secondary wall and-especially in plasmolyzed tubes-within the secondary wall of tube flanks and wall cap. The findings are discussed in connection with published schemes of membrane behaviour during exocytosis.Abbreviations EF extraplasmatic fracture face - IMP(s) intramembraneous particle(s) - PF plasmatic fracture face Extended version of a contribution (poster) presented at the 8th Int. Symp. on Sexual Reproduction in Seed Plants, Ferns and Mosses, Wageningen, The Netherlands, August 1984 Dedicated to Prof. Dr. H.F. Linskens (Nijmegen) on the occasion of his 65th birthday in 1986     

Role of HRB in clathrin-dependent endocytosis

Human immunodeficiency virus Rev-binding protein (HRB), also called human Rev-interacting protein (hRIP) or Rev/Rex activation domain binding (RAB) is a partner of the tyrosine kinase substrate EPS15, and it has been recovered in the AP-2 interactome. EPS15 and AP-2 are involved in endocytosis, but the function of HRB in this process is still unknown. Here we identified HRB as a partner of the vesicular SNARE tetanus neurotoxin-insensitive vesicle-associated membrane protein (TI-VAMP, also called VAMP7) in yeast two-hybrid screens and using biochemical assays. In HeLa cells, HRB localized both in the nucleus and in the cytoplasm. In the cytoplasm, HRB colocalized with clathrin-, AP-2-, EPS15-, and transferrin receptor-containing vesicles. We did not see significant colocalization between HRB and TI-VAMP in HeLa cells, and we saw partial colocalization with green fluorescent protein-TI-VAMP in stably expressing Madin-Darby canine kidney cells. Nevertheless using a pHLuorin-tagged TI-VAMP construct, we found that HRB and TI-VAMP colocalize close to the plasma membrane after 5 min of anti-green fluorescent protein antibody uptake. These results suggest that TI-VAMP and HRB may interact only during the early stages of endocytosis. Furthermore uptake experiments followed by fluorescence-activated cell sorting showed that the endocytosis of fluorescent transferrin and pHLuorin-TI-VAMP is strongly reduced in HRB knockdown cells. Altogether these results suggest that HRB is involved in clathrin-dependent endocytosis and recruits TI-VAMP in this process.     

A model for the mechanism of tip extension in pollen tubes

Three main mechanisms are proposed to account for the tip growth of pollen tubes. (1) The tip region is supported against the internal osmotic pressure of the cell by a fibrillar network, composed mainly of microfilaments, that is stabilized by calcium ions. Tip extension is promoted by a lowering of the local cytoplasmic calcium ion concentration, through uptake by the mitochondria and/or endoplasmic reticulum, which leads to a weakening of the fibrillar network. (2) Vesicles, derived from dictyosomes in the main body of the tube, fuse with the apical plasma membrane, providing new membrane and further carbohydrate for the wall. The rate of fusion is proportional to the rate of diffusion of calcium ion across the plasma membrane at the tip. (3) The callose lining present in the pollen tube wall, except at the tip, renders the wall impermeable and restricts entry of calcium ions to the apical plasma membrane. This restriction limits the rate of vesicle fusion, and tube growth, to the tip.This model is discussed in the light of previous observations on the growth and structure of pollen tubes under normal and experimental conditions.     

Enhanced clathrin-dependent endocytosis in the absence of calnexin

Background

Calnexin, together with calreticulin, constitute the calnexin/calreticulin cycle. Calnexin is a type I endoplasmic reticulum integral membrane protein and molecular chaperone responsible for the folding and quality control of newly-synthesized (glyco)proteins. The endoplasmic reticulum luminal domain of calnexin is responsible for lectin-like activity and interaction with nascent polypeptide chains. The role of the C-terminal, cytoplasmic portion of calnexin is not clear.

Methodology/Principal Findings

Using yeast two hybrid screen and immunoprecipitation techniques, we showed that the Src homology 3-domain growth factor receptor-bound 2-like (Endophilin) interacting protein 1 (SGIP1), a neuronal specific regulator of endocytosis, forms complexes with the C-terminal cytoplasmic domain of calnexin. The calnexin cytoplasmic C-tail interacts with SGIP1 C-terminal domains containing the adaptor complexes medium subunit (Adap-Comp-Sub) region. Calnexin-deficient cells have enhanced clathrin-dependent endocytosis in neuronal cells and mouse neuronal system. This is reversed by expression of full length calnexin or calnexin C-tail.

Conclusions/Significance

We show that the effects of SGIP1 and calnexin C-tail on clathrin-dependent endocytosis are due to modulation of the internalization of the receptor-ligand complexes. Enhanced clathrin-dependent endocytosis in the absence of calnexin may contribute to the neurological phenotype of calnexin-deficient mice.     

Growth and cellular organization of Arabidopsis pollen tubes in vitro

Tricellular pollen tubes of Arabidopsis thaliana were cultured in vitro on solid media and studied with respect to growth, cellular organization and ultrastructure, cytoskeletal organization, organelle movement, deposition and structure of the wall and the occurrence of coated pits, all elements assumed to be relevant for tip growth. For our ultrastructural studies we used freeze fixation and freeze substitution. Although Arabidopsis pollen tubes are broadly similar to those of bicellular species such as Nicotiana tabacum and Lilium spec. and in vivo grown pollen tubes of Arabidopsis, some differences occurred. The density of the equally distributed, relatively small (85 nm) secretory vesicles (SV) in the tip is low (five/µm ²). In between the SV of the tip, membranous material, possibly smooth endoplasmic reticulum, fragments of rough endoplasmic reticulum and loose ribosomes are present. The wall in the tip is not amorphous but layered and a secondary wall is formed already in the flanks of the tip. The general pattern of organelle motion is reverse fountain-like, but individual organelles move in distinct lanes at speeds of up to 2 µm/s, and about half of the organelle population shows a moderate velocity or Brownian movement. These properties are discussed in relation to the low growth rate (10 µm/h) of Arabidopsis pollen grown in vitro. The two similar sperm cells are closely attached and are always found near the vegetative nucleus. No surrounding wall and no cytoskeletal elements were obvious in the sperm cells. The preferential location of the mitochondria at the wall and the large (up to 400 nm) coated pits are unique for angiosperm pollen tubes. The size of the coated pits may allow not only membrane retrieval but also pinocytosis.     

Development of subprotoplasts from in vitro-grown tobacco pollen tubes

Summary Plasmolyzed pollen tubes of Nicotiana tabacum each released one to three subprotoplasts from their tips when treated with wall-degrading enzymes. Wall regeneration and the further development of the subprotoplasts were studied by both light and electron microscopy. Karyoplasts and cytoplasts incubated in a poor culture medium regenerated a cell wall within 60 min; cellulose microfibrils and callose were shown to be present. In 30%–40% of the cells, one-third of which were nucleate, cell chains and tubes developed by polar growth in a ratio of about 11. They sometimes reached a length 7–9 times the diameter of the former subprotoplast within 5 h of incubation. With longer incubation periods the cell wall became two-layered, its ultrastructure resembling that of the pollen tubes. The capacity of cytoplasts to regenerate a wall and develop cell chains and tubes can be explained by the properties ascribed to the cytoplasm of pollen tubes.Extended version of part of a contribution (poster) presented at the 14th International Botanical Congress, Berlin (West), July 1987.     

Fixation induces differential tip morphology and immunolocalization of the cytoskeleton in pollen tubes

The effects of chemical fixation on tip morphology and immunolocalization of the cytoskeleton in Tradescantia virginiana pollen tubes were evaluated using lour different fixation protocols differing in fixative type/concentration, fixation time, buffer system and additives. Apical regions were much more sensitive to fixation manipulations than more basipetal areas. The presence of the calcium chelator EGTA at 5 mM led to tip rupture and/or swelling in over 80% of germinated grains. However, low or no EGTA levels during fixation resulted in poor immunolocalizations, although lips had more normal morphology. Double fixation in which pollen tubes were first treated for a short period with higher fixative and lower EGTA (0.5 mM) concentrations, followed by lower fixative and higher EGTA (5.0 mM) concentrations, resulted in both improved preservation of pollen lube tip morphology and microfilament/microtubule localizations.     

Driving membrane curvature in clathrin-dependent and clathrin-independent endocytosis

Cellular activity depends to a large extent on membrane bilayer dynamics. Many processes, such as organelle biogenesis and vesicular transport, rely on alterations in membrane structure and shape. It is now widely accepted that intracellular membrane curvature generation and remodelling is mediated and regulated by protein action, and the mechanisms behind the processes are currently being revealed. Here, we will briefly discuss the key principles of membrane deformation and focus on different endocytic events that use various kinds of proteins to shape the plasma membrane into transport carriers. The entry routes are adopted to make sure that a vast variety of molecules on the cell surface can be regulated by endocytosis. The principles for membrane sculpting of endocytic carriers can be viewed either from a perspective of rigid coat budding or of flexible opportunistic budding. We will discuss these principles and their implications, focusing on clathrin-dependent and -independent carrier formation and the proteins involved in the respective pathways.     

Endocytosis in tobacco pollen tubes: visualisation and measurement of plasma membrane retrieval during different gravity conditions indicates gravity-dependence of endocytosis

We studied the effect of microgravity on endocytosis in growing tobacco pollen tubes by measuring the plasma membrane retrieval employing the fluorescent phospholipid bis-Bodipy FL C11- phosphatidylcholine as marker. Time course experiments under 1xg condition revealed a localised and relatively fast plasma membrane retrieval in the pollen tube tip region within the first minutes after lipid application. The rate of endocytotic bis-Bodipy FL C11- PC-modified plasma membrane retrieval is inhibited by hyper-g conditions achieved by centriftigal forces. In contrast, during the microgravity phase of a parabolic rocket flight the retrieval of the fluorescently-marked plasma membrane is distinctly enhanced. Our results show that microgravity exerts an unspecific physiological response in pollen tubes, most likely involving the cytoskeleton as inhibitor experiments indicate under 1xg condition.     

Distribution of microtubules during the growth of tobacco pollen tubes

Summary— The distribution of microtubules was investigated in Nicotiana tabacum pollen tubes at different stages of tube growth by immunofluorescence microscopy. Using specific antibodies, the presence of microtubules consisting of different tubulin isoforms was tested. α-, β- and tyrosinated α-tubulin were present within the tube, whereas the acetylated form was lacking. The presence of tubulin subunits in pollen tube extracts was also investigated by immunoblotting analyses. The use of a confocal laser scanning microscope integrated with computer-assisted imaging, allowed a detailed visualization of the microtubule distribution and organization. Cytoplasmic microtubules organized as short bundles with various orientations were detected at the apex of long tubes.     

The role of clathrin-dependent endocytosis in bacterial internalization

Internalization of bacteria into mammalian host cells has been studied extensively in the past two decades. These studies have highlighted the amazingly diverse strategies used by bacterial pathogens to induce their entry in non-phagocytic cells. The roles of actin and of the whole cytoskeletal machinery have been investigated in great detail for several invasive organisms, such as Salmonella, Shigella, Yersinia and Listeria. Recent results using Listeria highlight a role for the endocytosis machinery in bacterial entry, suggesting that clathrin-dependent endocytic mechanisms are also involved in internalization of large particles. This contrasts with the generally accepted dogma but agrees with previous studies of bacterial and viral infections and also of phagocytosis.