trans-Golgi Network Cisternae Research Articles

3D electron tomographic and biochemical analysis of ER, Golgi and trans Golgi network membrane systems in stimulated Venus flytrap (Dionaea muscipula) glandular cells

BackgroundThe insect-trapping leaves of Dionaea muscipula provide a model for studying the secretory pathway of an inducible plant secretory system. The leaf glands were induced with bovine serum albumin to secrete proteases that were characterized via zymogram activity gels over a 6-day period. The accompanying morphological changes of the endoplasmic reticulum (ER) and Golgi were analyzed using 3D electron tomography of glands preserved by high-pressure freezing/freeze substitution methods.ResultsSecretion of multiple cysteine and aspartic proteases occurred biphasically. The majority of the Golgi was organized in clusters consisting of 3–6 stacks surrounded by a cage-like system of ER cisternae. In these clusters, all Golgi stacks were oriented with their cis-most C1 cisterna facing an ER export site. The C1 Golgi cisternae varied in size and shape consistent with the hypothesis that they form de novo. Following induction, the number of ER-bound polysomes doubled, but no increase in COPII vesicles was observed. Golgi changes included a reduction in the number of cisternae per stack and a doubling of cisternal volume without increased surface area. Polysaccharide molecules that form the sticky slime cause swelling of the trans and trans Golgi network (TGN) cisternae. Peeling of the trans-most cisternae gives rise to free TGN cisternae. One day after gland stimulation, the free TGNs were frequently associated with loose groups of oriented actin-like filaments which were not seen in any other samples.ConclusionsThese findings suggest that the secretory apparatus of resting gland cells is “overbuilt” to enable the cells to rapidly up-regulate lytic enzyme production and secretion in response to prey trapping.

AP-1A controls secretory granule biogenesis and trafficking of membrane secretory granule proteins.

The adaptor protein 1A complex (AP-1A) transports cargo between the trans-Golgi network (TGN) and endosomes. In professional secretory cells, AP-1A also retrieves material from immature secretory granules (SGs). The role of AP-1A in SG biogenesis was explored using AtT-20 corticotrope tumor cells expressing reduced levels of the AP-1A μ1A subunit. A twofold reduction in μ1A resulted in a decrease in TGN cisternae and immature SGs and the appearance of regulated secretory pathway components in non-condensing SGs. Although basal secretion of endogenous SG proteins was unaffected, secretagogue-stimulated release was halved. The reduced μ1A levels interfered with the normal trafficking of carboxypeptidase D (CPD) and peptidylglycine α-amidating monooxygenase-1 (PAM-1), integral membrane enzymes that enter immature SGs. The non-condensing SGs contained POMC products and PAM-1, but not CPD. Based on metabolic labeling and secretion experiments, the cleavage of newly synthesized PAM-1 into PHM was unaltered, but PHM basal secretion was increased in sh-μ1A PAM-1 cells. Despite lacking a canonical AP-1A binding motif, yeast two-hybrid studies demonstrated an interaction between the PAM-1 cytosolic domain and AP-1A. Coimmunoprecipitation experiments with PAM-1 mutants revealed an influence of the luminal domains of PAM-1 on this interaction. Thus, AP-1A is crucial for normal SG biogenesis, function and composition.

Shrinkage and fragmentation of the trans-Golgi network in non-meristematic plant cells

Golgi products are exported from the trans-Golgi network (TGN) where they are sorted and packaged into secretory and clathrin-coated vesicles. We have examined TGN cisternae in Arabidopsis root columella cells and in maize basal endosperm transfer cells by electron microscopy/tomography. In these cell types, sizes of the TGN compartments decrease as they produce vesicles. After released from the Golgi, free TGN compartments continue to contract and they were seen to fragment into clusters of vesicles. The shrinkage of the plant TGN and its final disassembly suggest that the plant TGN is not a long-lasting organelle that is replenished regularly by membrane trafficking.

Electron Tomography of RabA4b‐ and PI‐4Kβ1‐Labeled Trans Golgi Network Compartments in Arabidopsis

The trans Golgi network (TGN) of plant cells sorts and packages Golgi products into secretory (SV) and clathrin-coated (CCV) vesicles. We have analyzed of TGN cisternae in Arabidopsis root meristem cells by cell fractionation and electron microscopy/tomography to establish reliable criteria for identifying TGN cisternae in plant cells, and to define their functional attributes. Transformation of a trans Golgi cisterna into a Golgi-associated TGN cisterna begins with cisternal peeling, the formation of SV buds outside the plane of the cisterna and a 30-35% reduction in cisternal membrane area. Free TGN compartments are defined as cisternae that have detached from the Golgi to become independent organelles. Golgi-associated and free TGN compartments, but not trans Golgi cisternae, bind anti-RabA4b and anti-phosphatidylinositol-4 kinase (PI-4K) antibodies. RabA4b and PI-4Kβ1 localize to budding SVs in the TGN and to SVs en route to the cell surface. SV and CCV release occurs simultaneously via cisternal fragmentation, which typically yields ∼30 vesicles and one to four residual cisternal fragments. Early endosomal markers, VHA-a1-green fluorescent protein (GFP) and SYP61-cyan fluorescent protein (CFP), colocalized with RabA4b in TGN cisternae, suggesting that the secretory and endocytic pathways converge at the TGN. pi4k1/pi4k2 knockout mutant plants produce SVs with highly variable sizes indicating that PI-4Kβ1/2 regulates SV size.

Tomographic evidence for continuous turnover of Golgi cisternae in Pichia pastoris.

The budding yeast Pichia pastoris contains ordered Golgi stacks next to discrete transitional endoplasmic reticulum (tER) sites, making this organism ideal for structure-function studies of the secretory pathway. Here, we have used P. pastoris to test various models for Golgi trafficking. The experimental approach was to analyze P. pastoris tER-Golgi units by using cryofixed and freeze-substituted cells for electron microscope tomography, immunoelectron microscopy, and serial thin section analysis of entire cells. We find that tER sites and the adjacent Golgi stacks are enclosed in a ribosome-excluding "matrix." Each stack contains three to four cisternae, which can be classified as cis, medial, trans, or trans-Golgi network (TGN). No membrane continuities between compartments were detected. This work provides three major new insights. First, two types of transport vesicles accumulate at the tER-Golgi interface. Morphological analysis indicates that the center of the tER-Golgi interface contains COPII vesicles, whereas the periphery contains COPI vesicles. Second, fenestrae are absent from cis cisternae, but are present in medial through TGN cisternae. The number and distribution of the fenestrae suggest that they form at the edges of the medial cisternae and then migrate inward. Third, intact TGN cisternae apparently peel off from the Golgi stacks and persist for some time in the cytosol, and these "free-floating" TGN cisternae produce clathrin-coated vesicles. These observations are most readily explained by assuming that Golgi cisternae form at the cis face of the stack, progressively mature, and ultimately dissociate from the trans face of the stack.

Architecture of the Golgi apparatus of a scale-forming alga: biogenesis and transport of scales.

Mechanisms of transport of secretory products across the Golgi apparatus (GA) as well as of scale formation in prymnesiophytes have remained controversial. We have used a quantitative morphological approach to study formation and transport of scales across the GA in haploid cells of Pleurochrysis sp. The GA of these cells differs from the GA of higher plants in at least six morphological characteristics. Our results show that scales form in the trans-Golgi network (TGN) and transit the TGN in heretofore unrecognized prosecretory vesicles. Prosecretory vesicles differentiate into secretory vesicles prior to exocytosis of scales to the cell surface. Because prosecretory vesicles are only fragments of TGN cisternae, the classical model of cisternal progression is not a valid mechanism of transport in this alga. TGN transport vesicles are also involved in scale formation; however, the role of tubular connections between cisternae of a single stack-TGN unit is not clear. The relationship of two morphological types of cisternal dilations to a membrane-associated, bottlebrush-shaped macromolecule of novel morphology suggests a new hypothesis for the biogenesis of scales.

Essential role played by the C-terminal domain of glycoprotein I in envelopment of varicella-zoster virus in the trans-Golgi network: interactions of glycoproteins with tegument.

Varicella-zoster virus (VZV) is enveloped in the trans-Golgi network (TGN). Here we report that glycoprotein I (gI) is required within the TGN for VZV envelopment. Enveloping membranous TGN cisternae were microscopically identified in cells infected with intact VZV. These sacs curved around, and ultimately enclosed, nucleocapsids. Tegument coated the concave face of these sacs, which formed the viral envelope, but the convex surface was tegument-free. TGN cisternae of cells infected with VZV mutants lacking gI (gI(Delta)) or its C (gI(DeltaC))- or N-terminal (gI(DeltaN))-terminal domains were uniformly tegument coated and adhered to one another, forming bizarre membranous stacks. Viral envelopment was compromised, and no virions were delivered to post-Golgi structures. The TGN was not gI-immunoreactive in cells infected with the gI(Delta) or gI(DeltaN) mutants, but it was in cells infected with gI(DeltaC) (because the ectodomains of gI and gE interact). The presence in the TGN of gI lacking a C-terminal domain, therefore, was not sufficient to maintain enveloping cisternae. In cells infected with intact VZV or with gI(Delta), gI(DeltaN), or gI(DeltaC) mutants, ORF10p immunoreactivity was concentrated on the cytosolic face of TGN membranes, suggesting that it interacts with the cytosolic domains of glycoproteins. Because of the gE-gI interaction, cotransfected cells that expressed gE or gI were able to target truncated forms of the other to the TGN. Our data suggest that the C-terminal domain of gI is required to segregate viral and cellular proteins in enveloping TGN cisternae.

Demonstration of acetylcholinesterase molecular forms in a continuous tubular lysosomal system of rat pancreatic acinar cells.

By applying the highly sensitive cytochemical Gautron's technique, we were able to reveal AChE activity in rat pancreatic acinar cells, particularly at the level of a complex membrane-bound network formed by tubules with varicosities located around the nuclei and close to the basolateral membrane. The Golgi apparatus was devoid of cytochemical reaction beside the trans-Golgi network cisternae, which showed a positive reaction. The RER of some acinar cells also presented a signal, demonstrating their capability of synthesizing AChE. Immunogold using a specific anti-AChE antibody yielded similar results. Double-labeling experiments corroborated the presence of enzyme cytochemical and immunocytochemical signals in the same lysosomal tubular network. Biochemical sedimentation assays confirmed the presence of AChE in acinar cells, which exists as two globular molecular forms, G(1) and G(4). These results were obtained with pancreatic tissue in situ as well as with isolated acinar cells maintained in culture and devoid of neural elements. The existence of a continuous tubular lysosomal network containing AChE is in agreement with previous reports on acinar and other cell types, and supports a more general hypothesis on dynamic continuities among cell structures. Whether AChE is being secreted by the acinar cells or internalized through this endo-lysosomal system was not defined. However, the capability of the acinar cells to synthesize AChE and to channel it through a tubular system is a good indication that the cells can modulate their cholinergic stimulation for optimal secretion of digestive enzymes.