Role of the Golgi Apparatus in the Blood-Brain Barrier: Golgi Protection May Be a Targeted Therapy for Neurological Diseases

The blood-brain barrier (BBB) protects the brain from toxic material in the blood, provides nutrients for brain tissues, and screens harmful substances from the brain. The specific brain microvascular endothelial cells (BMVECs), tight junction between endothelial cells, and astrocytes ensure proper function of the central nervous system (CNS). Pathological factors disrupt the integrity of the BBB by destroying the normal function of endothelial cells and decreasing the production of tight junction proteins or the expression of proteins specifically localized on astrocytes. Interestingly, fragmentation of the Golgi apparatus is observed in neurological diseases and is involved in the destruction of the BBB function. The Golgi acts as a processing center in which proteins are transported after being processed in the endoplasmic reticulum. Besides reprocessing, classifying, and packaging proteins, the Golgi apparatus (GA) also acts as a signaling platform and calcium pool. In this review, we summarized the current literature on the potential relationship between the Golgi and endothelial cells, tight junction, and astrocytes. The normal function of the BBB is maintained as long as the normal function and morphology of the GA are not disturbed. Furthermore, we speculate that protecting the Golgi may be a novel therapeutic approach to protect the BBB and treat neurological diseases due to BBB dysfunction.     

A Differential Staining Technique for Simultaneous Visualization of Mitotic Spindle and Chromosomes in Mammalian Cells

A new technique has been devised for staining the mitotic spindle in mammalian cells while preserving spindle structure and chromosome number. The cells are trypsinized and fixed with a 3:1 methanobacetic acid solution containing 4 mM MgCl₂ and 1.5 mM CaCl₂ at room temperature. The cells are then placed on slides and treated with 5% perchloric acid before staining with a 10% acetic acid solution containing safranin O and brilliant blue R. The preserved spindles appear dark blue against a light cytoplasmic background with chromosomes stained bright red. Individual chromosomes and chromatids are clearly visible. Positioning of the chromosomes relative to the spindle apparatus is readily ascertained allowing easy study of mitotic spindle and chromosome behavior.     

Electro-Acoustic Behavior of the Mitotic Spindle: A Semi-Classical Coarse-Grained Model

The regulation of chromosome separation during mitosis is not fully understood yet. Microtubules forming mitotic spindles are targets of treatment strategies which are aimed at (i) the triggering of the apoptosis or (ii) the interruption of uncontrolled cell division. Despite these facts, only few physical models relating to the dynamics of mitotic spindles exist up to now. In this paper, we present the first electromechanical model which enables calculation of the electromagnetic field coupled to acoustic vibrations of the mitotic spindle. This electromagnetic field originates from the electrical polarity of microtubules which form the mitotic spindle. The model is based on the approximation of resonantly vibrating microtubules by a network of oscillating electric dipoles. Our computational results predict the existence of a rapidly changing electric field which is generated by either driven or endogenous vibrations of the mitotic spindle. For certain values of parameters, the intensity of the electric field and its gradient reach values which may exert a not-inconsiderable force on chromosomes which are aligned in the spindle midzone. Our model may describe possible mechanisms of the effects of ultra-short electrical and mechanical pulses on dividing cells—a strategy used in novel methods for cancer treatment.     

A Role for Tlg1p in the Transport of Proteins within the Golgi Apparatus of Saccharomyces cerevisiae

Members of the syntaxin protein family participate in the docking-fusion step of several intracellular vesicular transport events. Tlg1p has been identified as a nonessential protein required for efficient endocytosis as well as the maintenance of normal levels of trans-Golgi network proteins. In this study we independently describe Tlg1p as an essential protein required for cell viability. Depletion of Tlg1p in vivo causes a defect in the transport of the vacuolar protein carboxypeptidase Y through the early Golgi. Temperature-sensitive (ts) mutants of Tlg1p also accumulate the endoplasmic reticulum/cis-Golgi form of carboxypeptidase Y at the nonpermissive temperature (38 degrees C) and exhibit underglycosylation of secreted invertase. Overexpression of Tlg1p complements the growth defect of vti1-11 at the nonpermissive temperature, whereas incomplete complementation was observed with vti1-1, further suggesting a role for Tlg1p in the Golgi apparatus. Overexpression of Sed5p decreases the viability of tlg1 ts mutants compared with wild-type cells, suggesting that tlg1 ts mutants are more susceptible to elevated levels of Sed5p. Tlg1p is able to bind His6-tagged Sec17p (yeast alpha-SNAP) in a dose-dependent manner and enters into a SNARE complex with Vti1p, Tlg2p, and Vps45p. Morphological analyses by electron microscopy reveal that cells depleted of Tlg1p or tlg1 ts mutants incubated at the restrictive temperature accumulate 40- to 50-nm vesicles and experience fragmentation of the vacuole.     

Study of GOLPH3: a Potential Stress-Inducible Protein from Golgi Apparatus

Although the Golgi apparatus has been studied extensively for over 100 years, the complex structure-function relationships have yet to be elucidated. It is well known that the Golgi complex plays an important role in the transport, processing, sorting, and targeting of numerous proteins and lipids destined for secretion, plasma membrane, and lysosomes. Increasing evidence suggests that the Golgi apparatus is a sensor and common downstream effector of stress signals in cell death pathways. It undergoes disassembly and fragmentation in several neurological disorders. Recent studies indicate that Golgi phosphoprotein 3 (GOLPH3 also known as GPP34/GMx33/MIDAS), a peripheral membrane protein of trans-Golgi network, represents an exciting new class of oncoproteins involved in cell signal transduction and is potentially mobilized by stress. In this review, we focus on the importance of GOLPH3 in vesicular trafficking, Golgi architecture maintenance, receptor sorting, protein glycosylation, and further discuss its potential in signal sensing in stress response.     

Schistosoma mansoni: cytochemistry and morphology of the gastrodermal Golgi Apparatus

The gastrodermal Golgi apparatus of adult Schistosoma mansoni displays two distinct morphologies. In one type, there is an identifiable cis (forming) face where vesicles from the endoplasmic reticulum fuse to form the cisternae. A morphological change occurs in the cisternae as the trans (emitting) face is approached with the cisternae becoming progressively flattened. The cisternae at the emitting face produce a membrane-bound secretory granule with moderately electron-dense contents and a vacuolar structure that may be analogous to a condensing vacuole as reported in several vertebrate secretory cells. In a second type, vesicles possessing a thicker membrane than those of the transfer vesicles are observed at the emitting face. They are not observed when the secretory granules are present. Several cytochemical markers were used to aid in studying the polarity of the Golgi apparatus. Enzymes studied were thiamine pyrophosphatase (TPPase) (EC 3.6.1.1), nucleoside diphosphatase (NDPase) (EC 3.6.1.6) using uridine diphosphate as a substrate, and nicotinamide adenine dinucleotide phosphatase (NADPase) (EC 3.1.3.2). Reaction products from all enzyme markers were observed in the cisternae and, to some extent, in the transfer vesicles. At times, NADPase and TPPase reaction products were observed in all cisternae and in the transfer vesicles of the Golgi. When this distribution was evident, the latter vesicles were observed in clusters occasionally fusing with lipid-like globules dispersed throughout the gastrodermis. Heterogeneity in cisternae was observed when NDPase, TPPase, and osmium reduction techniques were used. NDPase activity was limited to the middle cisternae while reduced osmium was observed in the outer two cisternae and in some transfer vesicles. TPPase reaction product was also observed in the secretory granules and in the condensing vacuoles. It is hypothesized that a functional bipolarity may be demonstrated by the Golgi. Under certain stress conditions, the forming face of the Golgi may package lysosomal enzymes while the emitting region of the Golgi appears to be responsible for the packaging of the secretory granules. The fusion of transfer vesicles and, at times, secretory granules with lipid-like globules is postulated to represent a mechanism by which enzymes may be transported to the lumen of the cecum.     

Asymmetry of the Budding Yeast Tem1 GTPase at Spindle Poles Is Required for Spindle Positioning But Not for Mitotic Exit

The asymmetrically dividing yeast S. cerevisiae assembles a bipolar spindle well after establishing the future site of cell division (i.e., the bud neck) and the division axis (i.e., the mother-bud axis). A surveillance mechanism called spindle position checkpoint (SPOC) delays mitotic exit and cytokinesis until the spindle is properly positioned relative to the mother-bud axis, thereby ensuring the correct ploidy of the progeny. SPOC relies on the heterodimeric GTPase-activating protein Bub2/Bfa1 that inhibits the small GTPase Tem1, in turn essential for activating the mitotic exit network (MEN) kinase cascade and cytokinesis. The Bub2/Bfa1 GAP and the Tem1 GTPase form a complex at spindle poles that undergoes a remarkable asymmetry during mitosis when the spindle is properly positioned, with the complex accumulating on the bud-directed old spindle pole. In contrast, the complex remains symmetrically localized on both poles of misaligned spindles. The mechanism driving asymmetry of Bub2/Bfa1/Tem1 in mitosis is unclear. Furthermore, whether asymmetry is involved in timely mitotic exit is controversial. We investigated the mechanism by which the GAP Bub2/Bfa1 controls GTP hydrolysis on Tem1 and generated a series of mutants leading to constitutive Tem1 activation. These mutants are SPOC-defective and invariably lead to symmetrical localization of Bub2/Bfa1/Tem1 at spindle poles, indicating that GTP hydrolysis is essential for asymmetry. Constitutive tethering of Bub2 or Bfa1 to both spindle poles impairs SPOC response but does not impair mitotic exit. Rather, it facilitates mitotic exit of MEN mutants, likely by increasing the residence time of Tem1 at spindle poles where it gets active. Surprisingly, all mutant or chimeric proteins leading to symmetrical localization of Bub2/Bfa1/Tem1 lead to increased symmetry at spindle poles of the Kar9 protein that mediates spindle positioning and cause spindle misalignment. Thus, asymmetry of the Bub2/Bfa1/Tem1 complex is crucial to control Kar9 distribution and spindle positioning during mitosis.     

Asymmetric Segregation and Polarized Redistribution of Pole Plasm During Early Cleavages in the Tubifex Embryo: Role of Actin Networks and Mitotic Apparatus

In the precleavage zygote of Tubifex , pole plasm, which is yolk-free cytoplasm, is located at the animal and vegetal poles. The present study describes the fate and localization pattern of the pole plasms in embryonic development of Tubifex . The process of pole plasm localization during cleavage stages is comprised of three steps. The first step is asymmetric segregation which results in bipolar localization of pole plasm masses in the D-cell of the 4-cell embryo. The spatial organization of pole plasm at this stage depends on F-actin but not on microtubules. The second step is the redistribution of the vegetal pole plasm toward the animal pole and its unification with the animal pole plasm. These give rise to localization of unified pole plasm at the animal side (i.e. future dorsal side of the embryo) of the D-quadrant. The polarized redistribution is sensitive to colchicine and topographically related to the mitotic apparatus located at the animal pole of the D-cell. Electron microscopy shows the association with astral microtubules of constituents of pole plasm, suggesting the involvement of astral microtubules in cytoplasmic movement which gives rise to redistribution. In addition, centrifuge experiments suggest that the directional information for this polarized redistribution may be provided by some cytoplasmic organizations which are resistant to centrifugal force. The last step of the localization process is partitioning of unified pole plasm into two micromeres 2d and 4d. The spatial organization of pole plasm at this stage depends on microtubules but not on F-actin.     

UDP-GlcNAc:Glycoprotein GlcNAc-Transferase is Located in the Golgi Apparatus of Developing Bean Cotyledons

The transport and accumulation of phytohemagglutinin in developing bean (Phaseolus vulgaris L.) cotyledons is accompanied by the transient presence of N-acetylglucosamine (GlcNAc) residues on the oligosaccharide sidechains of this glycoprotein. These peripheral GlcNAc residues can be distinguished from those in the chitobiose portion of the oligosaccharide sidechains by their sensitivity to removal by the exoglycosidase β-N-acetylglucosaminidase. GlcNAc residues sensitive to removal by β-N-acetylglucosaminidase are present not only on phytohemagglutinin, but also on other newly synthesized proteins. The enzyme UDPGlcNAc:glycoprotein GlcNAc-transferase which transfers GlcNAc residues to glycoproteins was first described by Davies and Delmer (Plant Physiol 1981 68: 284-291). The data presented here show that this enzyme is associated with the Golgi complex of developing cotyledons.     

Cohesion Fatigue Explains Why Pharmacological Inhibition of the APC/C Induces a Spindle Checkpoint-Dependent Mitotic Arrest

The Spindle Assembly Checkpoint (SAC) delays the onset of anaphase in response to unattached kinetochores by inhibiting the activity of the Anaphase-Promoting Complex/Cyclosome (APC/C), an E3 ubiquitin ligase. Once all the chromosomes have bioriented, SAC signalling is somehow silenced, which allows progression through mitosis. Recent studies suggest that the APC/C itself participates in SAC silencing by targeting an unknown factor for proteolytic degradation. Key evidence in favour of this model comes from the use of proTAME, a small molecule inhibitor of the APC/C. In cells, proTAME causes a mitotic arrest that is SAC-dependent. Even though this observation comes at odds with the current view that the APC/C acts downstream of the SAC, it was nonetheless argued that these results revealed a role for APC/C activity in SAC silencing. However, we show here that the mitotic arrest induced by proTAME is due to the induction of cohesion fatigue, a phenotype that is caused by the loss of sister chromatid cohesion following a prolonged metaphase. Under these conditions, the SAC is re-activated and APC/C inhibition is maintained independently of proTAME. Therefore, these results provide a simpler explanation for why the proTAME-induced mitotic arrest is also dependent on the SAC. While these observations question the notion that the APC/C is required for SAC silencing, we nevertheless show that APC/C activity does partially contribute to its own release from inhibitory complexes, and importantly, this does not depend on proteasome-mediated degradation.     

Spindle asymmetry: a compass for the cell

Spatial coordination of the cell-division axis with cellular polarity and/or with the position of neighboring cells is crucial for embryonic development, organogenesis and tissue homeostasis. In most cell types, the position of the mitotic spindle at the onset of anaphase dictates the orientation of the division axis; in unicellular organisms, it plays an important role in chromosome segregation. Cortical factors play a key role in the orientation of the spindle. Recent data from yeast reveal that the spindle does not passively react to cortical signals but actively interprets them to find its correct position. We review the data leading to a "compass model" for spindle positioning and discuss its potential generality.     

A Model for the Self-Organization of Vesicular Flux and Protein Distributions in the Golgi Apparatus

The generation of two non-identical membrane compartments via exchange of vesicles is considered to require two types of vesicles specified by distinct cytosolic coats that selectively recruit cargo, and two membrane-bound SNARE pairs that specify fusion and differ in their affinities for each type of vesicles. The mammalian Golgi complex is composed of 6–8 non-identical cisternae that undergo gradual maturation and replacement yet features only two SNARE pairs. We present a model that explains how distinct composition of Golgi cisternae can be generated with two and even a single SNARE pair and one vesicle coat. A decay of active SNARE concentration in aging cisternae provides the seed for a cistrans SNARE gradient that generates the predominantly retrograde vesicle flux which further enhances the gradient. This flux in turn yields the observed inhomogeneous steady-state distribution of Golgi enzymes, which compete with each other and with the SNAREs for incorporation into transport vesicles. We show analytically that the steady state SNARE concentration decays exponentially with the cisterna number. Numerical solutions of rate equations reproduce the experimentally observed SNARE gradients, overlapping enzyme peaks in cis, medial and trans and the reported change in vesicle nature across the Golgi: Vesicles originating from younger cisternae mostly contain Golgi enzymes and SNAREs enriched in these cisternae and extensively recycle through the Endoplasmic Reticulum (ER), while the other subpopulation of vesicles contains Golgi proteins prevalent in older cisternae and hardly reaches the ER.     

Recycling of Golgi-resident Glycosyltransferases through the ER Reveals a Novel Pathway and Provides an Explanation for Nocodazole-induced Golgi Scattering

During microtubule depolymerization, the central, juxtanuclear Golgi apparatus scatters to multiple peripheral sites. We have tested here whether such scattering is due to a fragmentation process and subsequent outward tracking of Golgi units or if peripheral Golgi elements reform through a novel recycling pathway. To mark the Golgi in HeLa cells, we stably expressed the Golgi stack enzyme N-acetylgalactosaminyltransferase-2 (GalNAc-T2) fused to the green fluorescent protein (GFP) or to an 11–amino acid epitope, VSV-G (VSV), and the trans/TGN enzyme β1,4-galactosyltransferase (GalT) fused to GFP. After nocodazole addition, time-lapse microscopy of GalNAc-T2–GFP and GalT–GFP revealed that scattered Golgi elements appeared abruptly and that no Golgi fragments tracked outward from the compact, juxtanuclear Golgi complex. Once formed, the scattered structures were relatively stable in fluorescence intensity for tens of minutes. During the entire process of dispersal, immunogold labeling for GalNAc-T2–VSV and GalT showed that these were continuously concentrated over stacked Golgi cisternae and tubulovesicular Golgi structures similar to untreated cells, suggesting that polarized Golgi stacks reform rapidly at scattered sites. In fluorescence recovery after photobleaching over a narrow (FRAP) or wide area (FRAP-W) experiments, peripheral Golgi stacks continuously exchanged resident proteins with each other through what appeared to be an ER intermediate. That Golgi enzymes cycle through the ER was confirmed by microinjecting the dominant-negative mutant of Sar1 (Sar1p^dn) blocking ER export. Sar1p^dn was either microinjected into untreated or nocodazole-treated cells in the presence of protein synthesis inhibitors. In both cases, this caused a gradual accumulation of GalNAc-T2–VSV in the ER. Few to no peripheral Golgi elements were seen in the nocodazole-treated cells microinjected with Sar1p^dn. In conclusion, we have shown that Golgi-resident glycosylation enzymes recycle through the ER and that this novel pathway is the likely explanation for the nocodazole-induced Golgi scattering observed in interphase cells.     

Fission Yeast Bub1 Is a Mitotic Centromere Protein Essential for the Spindle Checkpoint and the Preservation of Correct Ploidy through Mitosis

The spindle checkpoint ensures proper chromosome segregation by delaying anaphase until all chromosomes are correctly attached to the mitotic spindle. We investigated the role of the fission yeast bub1 gene in spindle checkpoint function and in unperturbed mitoses. We find that bub1 ⁺ is essential for the fission yeast spindle checkpoint response to spindle damage and to defects in centromere function. Activation of the checkpoint results in the recruitment of Bub1 to centromeres and a delay in the completion of mitosis. We show that Bub1 also has a crucial role in normal, unperturbed mitoses. Loss of bub1 function causes chromosomes to lag on the anaphase spindle and an increased frequency of chromosome loss. Such genomic instability is even more dramatic in Δbub1 diploids, leading to massive chromosome missegregation events and loss of the diploid state, demonstrating that bub1 ⁺ function is essential to maintain correct ploidy through mitosis. As in larger eukaryotes, Bub1 is recruited to kinetochores during the early stages of mitosis. However, unlike its vertebrate counterpart, a pool of Bub1 remains centromere-associated at metaphase and even until telophase. We discuss the possibility of a role for the Bub1 kinase after the metaphase–anaphase transition.