Deciphering the sub-Golgi localization of glycosyltransferases via 3D super-resolution imaging.

The Golgi apparatus, a crucial organelle involved in protein processing, including glycosylation, exhibits complex sub-structures, i.e., cis-, medial, and trans-cisternae. This study investigated the distribution of glycosyltransferases within the Golgi apparatus of mammalian cells via 3D super-resolution imaging. Focusing on human glycosyltransferases involved in N-glycan modification, we found that even enzymes presumed to coexist in the same Golgi compartment exhibit nuanced variations in localization. By artificially making their N-terminal regions [composed of a cytoplasmic, transmembrane, and stem segment (CTS)] identical, it was possible to enhance the degree of their colocalization, suggesting the decisive role of this region in determining the sub-Golgi localization of enzymes. Ultimately, this study reveals the molecular codes within CTS regions as key determinants of glycosyltransferase localization, providing insights into precise control over the positioning of glycosyltransferases, and consequently, the interactions between glycosyltransferases and substrate glycoproteins as cargoes in the secretory pathway. This study advances our understanding of Golgi organization and opens avenues for programming the glycosylation of proteins for clinical applications.Key words: Golgi apparatus, glycosyltransferase, 3D super-resolution imaging, N-glycosylation.

Extremely high spatiotemporal resolution microscopy for live cell imaging by single photon counting, noise elimination, and a novel restoration algorithm based on probability calculation.

Optical microscopy is essential for direct observation of dynamic phenomena in living cells. According to the classic optical theories, the images obtained through light microscopes are blurred for about half the wavelength of light, and therefore small structures below this "diffraction limit" were thought unresolvable by conventional optical microscopy. In reality, accurately obtained optical images contain complete information about the observed objects. Temporal resolution is also important for the observation of dynamic phenomena. A challenge exists here to overcome the trade-off between the time required for measurement and the accuracy of the measurement. The present paper describes a concrete methodology for reconstructing the structure of an observed object, based on the information contained in the image obtained by optical microscopy. It is realized by accurate single photon counting, complete noise elimination, and a novel restoration algorithm based on probability calculation. This method has been implemented in the Super-resolution Confocal Live Imaging Microscopy (SCLIM) we developed. The new system named SCLIM2M achieves unprecedented high spatiotemporal resolution. We have succeeded in capturing sub-diffraction-limit structures with millisecond-level dynamics of organelles and vesicles in living cells, which were never observed by conventional optical microscopy. Actual examples of the high-speed and high-resolution 4D observation of living cells are presented.

Dynamic movement of the Golgi unit and its glycosylation enzyme zones.

Knowledge on the distribution and dynamics of glycosylation enzymes in the Golgi is essential for better understanding this modification. Here, using a combination of CRISPR/Cas9 knockin technology and super-resolution microscopy, we show that the Golgi complex is assembled by a number of small 'Golgi units' that have 1-3 µm in diameter. Each Golgi unit contains small domains of glycosylation enzymes which we call 'zones'. The zones of N- and O-glycosylation enzymes are colocalised. However, they are less colocalised with the zones of a glycosaminoglycan synthesizing enzyme. Golgi units change shapes dynamically and the zones of glycosylation enzymes rapidly move near the rim of the unit. Photobleaching analysis indicates that a glycosaminoglycan synthesizing enzyme moves between units. Depletion of giantin dissociates units and prevents the movement of glycosaminoglycan synthesizing enzymes, which leads to insufficient glycosaminoglycan synthesis. Thus, we show the structure-function relationship of the Golgi and its implications in human pathogenesis.

Spatiotemporal dissection of the Golgi apparatus and the ER-Golgi intermediate compartment in budding yeast.

Cargo traffic through the Golgi apparatus is mediated by cisternal maturation, but it remains largely unclear how the cis-cisternae, the earliest Golgi sub-compartment, is generated and how the Golgi matures into the trans-Golgi network (TGN). Here, we use high-speed and high-resolution confocal microscopy to analyze the spatiotemporal dynamics of a diverse set of proteins that reside in and around the Golgi in budding yeast. We find many mobile punctate structures that harbor yeast counterparts of mammalian endoplasmic reticulum (ER)-Golgi intermediate compartment (ERGIC) proteins, which we term 'yeast ERGIC'. It occasionally exhibits approach and contact behavior toward the ER exit sites and gradually matures into the cis-Golgi. Upon treatment with the Golgi-disrupting agent brefeldin A, the ERGIC proteins form larger aggregates corresponding to the Golgi entry core compartment in plants, while cis- and medial-Golgi proteins are absorbed into the ER. We further analyze the dynamics of several late Golgi proteins to better understand the Golgi-TGN transition. Together with our previous studies, we demonstrate a detailed spatiotemporal profile of the entire cisternal maturation process from the ERGIC to the Golgi and further to the TGN.

Golgi retention and oncogenic KIT signaling via PLCγ2-PKD2-PI4KIIIß activation in gastrointestinal stromal tumor cells.

Most gastrointestinal stromal tumors (GISTs) develop due to gain-of-function mutations in the tyrosine kinase gene, KIT. We recently showed that mutant KIT mislocalizes to the Golgi area and initiates uncontrolled signaling. However, the molecular mechanisms underlying its Golgi retention remain unknown. Here, we show that protein kinase D2 (PKD2) is activated by the mutant, which causes Golgi retention of KIT. In PKD2-inhibited cells, KIT migrates from the Golgi region to lysosomes and subsequently undergoes degradation. Importantly, delocalized KIT cannot trigger downstream activation. In the Golgi/trans-Golgi network (TGN), KIT activates the PKD2-phosphatidylinositol 4-kinase IIIß (PKD2-PI4KIIIß) pathway through phospholipase Cγ2 (PLCγ2) to generate a PI4P-rich membrane domain, where the AP1-GGA1 complex is aberrantly recruited. Disruption of any factors in this cascade results in the release of KIT from the Golgi/TGN. Our findings show the molecular mechanisms underlying KIT mislocalization and provide evidence for a strategy for inhibition of oncogenic signaling.

The yeast endocytic early/sorting compartment exists as an independent sub-compartment within the trans-Golgi network.

Although budding yeast has been extensively used as a model organism for studying organelle functions and intracellular vesicle trafficking, whether it possesses an independent endocytic early/sorting compartment that sorts endocytic cargos to the endo-lysosomal pathway or the recycling pathway has long been unclear. The structure and properties of the endocytic early/sorting compartment differ significantly between organisms; in plant cells, the trans-Golgi network (TGN) serves this role, whereas in mammalian cells a separate intracellular structure performs this function. The yeast syntaxin homolog Tlg2p, widely localizing to the TGN and endosomal compartments, is presumed to act as a Q-SNARE for endocytic vesicles, but which compartment is the direct target for endocytic vesicles remained unanswered. Here we demonstrate by high-speed and high-resolution 4D imaging of fluorescently labeled endocytic cargos that the Tlg2p-residing compartment within the TGN functions as the early/sorting compartment. After arriving here, endocytic cargos are recycled to the plasma membrane or transported to the yeast Rab5-residing endosomal compartment through the pathway requiring the clathrin adaptors GGAs. Interestingly, Gga2p predominantly localizes at the Tlg2p-residing compartment, and the deletion of GGAs has little effect on another TGN region where Sec7p is present but suppresses dynamics of the Tlg2-residing early/sorting compartment, indicating that the Tlg2p- and Sec7p-residing regions are discrete entities in the mutant. Thus, the Tlg2p-residing region seems to serve as an early/sorting compartment and function independently of the Sec7p-residing region within the TGN.

Super-Resolution Live Imaging of Cargo Traffic Through the Golgi Apparatus in Mammalian Cells.

Super-resolution confocal live imaging microscopy (SCLIM) we developed provides high-speed, high-resolution, three- and four-dimensional, and multicolor simultaneous imaging. Using this technology, we are now able to observe the fine details of various dynamic events going on in living cells, such as membrane traffic and organelle dynamics. The retention using selective hooks (RUSH) system is a powerful tool to control synchronous release of natural cargo proteins of interest from the endoplasmic reticulum in mammalian cells. In this chapter, we describe a method for visualizing secretory cargo traffic within and around the Golgi apparatus in HeLa cells using SCLIM in combination with the RUSH assay.

Spatiotemporal dissection of the trans-Golgi network in budding yeast.

The trans-Golgi network (TGN) acts as a sorting hub for membrane traffic. It receives newly synthesized and recycled proteins, and sorts and delivers them to specific targets such as the plasma membrane, endosomes and lysosomes/vacuoles. Accumulating evidence suggests that the TGN is generated from the trans-most cisterna of the Golgi by maturation, but the detailed transition processes remain obscure. Here, we examine spatiotemporal assembly dynamics of various Golgi/TGN-resident proteins in budding yeast by high-speed and high-resolution spinning-disk confocal microscopy. The Golgi-TGN transition gradually proceeds via at least three successive stages: the 'Golgi stage' where glycosylation occurs; the 'early TGN stage', which receives retrograde traffic; and the 'late TGN stage', where transport carriers are produced. During the stage transition periods, earlier and later markers are often compartmentalized within a cisterna. Furthermore, for the late TGN stage, various types of coat/adaptor proteins exhibit distinct assembly patterns. Taken together, our findings characterize the identity of the TGN as a membrane compartment that is structurally and functionally distinguishable from the Golgi.This article has an associated First Person interview with the first author of the paper.

Visualization of Clathrin-Mediated Endocytosis During Semaphorin-Guided Axonal Growth.

Semaphorin3A (Sema3A) guides axonal growth during neuronal network development. Accumulating evidence indicates that Sema3A-induced growth cone collapse and repulsion involve endocytic membrane trafficking in the growth cone. It is now possible to visualize endocytic processes in living cells using total internal reflection fluorescence microscopy (TIRFM), a powerful tool for imaging dynamic subcellular events at the plasma membrane. In this chapter, we describe a method for TIRFM observation and analysis of clathrin-mediated endocytosis in growth cones of chicken dorsal root ganglion neurons that receive an extracellular concentration gradient of Sema3A in a culture medium.

Cyclic Nucleotide Control of Microtubule Dynamics for Axon Guidance.

UNLABELLED: Graded distribution of intracellular second messengers, such as Ca(2+) and cyclic nucleotides, mediates directional cell migration, including axon navigational responses to extracellular guidance cues, in the developing nervous system. Elevated concentrations of cAMP or cGMP on one side of the neuronal growth cone induce its attractive or repulsive turning, respectively. Although effector processes downstream of Ca(2+) have been extensively studied, very little is known about the mechanisms that enable cyclic nucleotides to steer migrating cells. Here, we show that asymmetric cyclic nucleotide signaling across the growth cone mediates axon guidance via modulating microtubule dynamics and membrane organelle transport. In embryonic chick dorsal root ganglion neurons in culture, contact of an extending microtubule with the growth cone leading edge induces localized membrane protrusion at the site of microtubule contact. Such a contact-induced protrusion requires exocytosis of vesicle-associated membrane protein 7 (VAMP7)-positive vesicles that have been transported centrifugally along the microtubule. We found that the two cyclic nucleotides counteractively regulate the frequency of microtubule contacts and targeted delivery of VAMP7 vesicles: cAMP stimulates and cGMP inhibits these events, thereby steering the growth cone in the opposite directions. By contrast, Ca(2+) signals elicit no detectable change in either microtubule contacts or VAMP7 vesicle delivery during Ca(2+)-induced growth cone turning. Our findings clearly demonstrate growth cone steering machinery downstream of cyclic nucleotide signaling and highlight a crucial role of dynamic microtubules in leading-edge protrusion for cell chemotaxis. SIGNIFICANCE STATEMENT: Developing neurons can extend long axons toward their postsynaptic targets. The tip of each axon, called the growth cone, recognizes extracellular guidance cues and navigates the axon along the correct path. Here we show that asymmetric cyclic nucleotide signaling across the growth cone mediates axon guidance through localized regulation of microtubule dynamics and resulting recruitment of specific populations of membrane vesicles to the growth cone's leading edge. Remarkably, cAMP stimulates microtubule growth and membrane protrusion, whereas cGMP promotes microtubule retraction and membrane senescence, explaining the opposite directional polarities of growth cone turning induced by these cyclic nucleotides. This study reveals a novel microtubule-based mechanism through which cyclic nucleotides polarize the growth cone steering machinery for bidirectional axon guidance.

Exocytic and endocytic membrane trafficking in axon development.

In the complex neuronal circuits in the nervous systems, billions of neurons are precisely interconnected by long, thin processes called the axons. The growth cone, a highly motile structure at the tip of an extending axon, navigates by responding to a variety of extracellular molecular cues toward their distant target cells and make synaptic connections. Emerging evidence indicates that exocytic and endocytic membrane trafficking systems play multiple important roles in the regulation of such axonal morphogenetic processes. Exocytosis and endocytosis organize the subcellular distribution of membrane-associated molecules, such as receptors, cell adhesion molecules, and cytoskeletal regulators, to control intracellular signaling and driving machineries. Furthermore, the exocytosis of trophic factors and extracellular proteinases act on surrounding microenvironments to affect growth cone motility. In this Review Article, we summarize our current understanding of the regulation and function of exocytic and endocytic membrane trafficking in axon morphogenesis during development, and discuss potential mechanisms of how the membrane trafficking systems exert such morphological changes.

Steering neuronal growth cones by shifting the imbalance between exocytosis and endocytosis.

Extracellular molecular cues guide migrating growth cones along specific routes during development of axon tracts. Such processes rely on asymmetric elevation of cytosolic Ca(2+) concentrations across the growth cone that mediates its attractive or repulsive turning toward or away from the side with Ca(2+) elevation, respectively. Downstream of these Ca(2+) signals, localized activation of membrane trafficking steers the growth cone bidirectionally, with endocytosis driving repulsion and exocytosis causing attraction. However, it remains unclear how Ca(2+) can differentially regulate these opposite membrane-trafficking events. Here, we show that growth cone turning depends on localized imbalance between exocytosis and endocytosis and identify Ca(2+)-dependent signaling pathways mediating such imbalance. In embryonic chicken dorsal root ganglion neurons, repulsive Ca(2+) signals promote clathrin-mediated endocytosis through a 90 kDa splice variant of phosphatidylinositol-4-phosphate 5-kinase type-1γ (PIPKIγ90). In contrast, attractive Ca(2+) signals facilitate exocytosis but suppress endocytosis via Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) and cyclin-dependent kinase 5 (Cdk5) that can inactivate PIPKIγ90. Blocking CaMKII or Cdk5 leads to balanced activation of both exocytosis and endocytosis that causes straight growth cone migration even in the presence of guidance signals, whereas experimentally perturbing the balance restores the growth cone's turning response. Remarkably, the direction of this resumed turning depends on relative activities of exocytosis and endocytosis, but not on the type of guidance signals. Our results suggest that navigating growth cones can be redirected by shifting the imbalance between exocytosis and endocytosis, highlighting the importance of membrane-trafficking imbalance for axon guidance and, possibly, for polarized cell migration in general.

Paxillin phosphorylation counteracts proteoglycan-mediated inhibition of axon regeneration.

In the adult central nervous system, the tips of axons severed by injury are commonly transformed into dystrophic endballs and cease migration upon encountering a rising concentration gradient of inhibitory proteoglycans. However, intracellular signaling networks mediating endball migration failure remain largely unknown. Here we show that manipulation of protein kinase A (PKA) or its downstream adhesion component paxillin can reactivate the locomotive machinery of endballs in vitro and facilitate axon growth after injury in vivo. In dissociated cultures of adult rat dorsal root ganglion neurons, PKA is activated in endballs formed on gradients of the inhibitory proteoglycan aggrecan, and pharmacological inhibition of PKA promotes axon growth on aggrecan gradients most likely through phosphorylation of paxillin at serine 301. Remarkably, pre-formed endballs on aggrecan gradients resume forward migration in response to PKA inhibition. This resumption of endball migration is associated with increased turnover of adhesive point contacts dependent upon paxillin phosphorylation. Furthermore, expression of phosphomimetic paxillin overcomes aggrecan-mediated growth arrest of endballs, and facilitates axon growth after optic nerve crush in vivo. These results point to the importance of adhesion dynamics in restoring endball migration and suggest a potential therapeutic target for axon tract repair.

Intracellular signaling and membrane trafficking control bidirectional growth cone guidance.

The formation of precise neuronal networks is critically dependent on the motility of axonal growth cones. Extracellular gradients of guidance cues evoke localized Ca(2+) elevations to attract or repel the growth cone. Recent studies strongly suggest that the polarity of growth cone guidance, with respect to the localization of Ca(2+) signals, is determined by Ca(2+) release from the endoplasmic reticulum (ER) in the following manner: Ca(2+) signals containing ER Ca(2+) release cause growth cone attraction, while Ca(2+) signals without ER Ca(2+) release cause growth cone repulsion. Recent studies have also shown that exocytic and endocytic membrane trafficking can drive growth cone attraction and repulsion, respectively, downstream of Ca(2+) signals. Most likely, these two mechanisms underlie cue-induced axon guidance, in which a localized imbalance between exocytosis and endocytosis dictates bidirectional growth cone steering. In this Update Article, I summarize recent advances in growth cone research and propose that polarized membrane trafficking plays an instructive role to spatially localize steering machineries, such as cytoskeletal components and adhesion molecules.

Second messengers and membrane trafficking direct and organize growth cone steering.

Graded distributions of extracellular cues guide developing axons toward their targets. A network of second messengers - Ca(2+) and cyclic nucleotides - shapes cue-derived information into either attractive or repulsive signals that steer growth cones bidirectionally. Emerging evidence suggests that such guidance signals create a localized imbalance between exocytosis and endocytosis, which in turn redirects membrane, adhesion and cytoskeletal components asymmetrically across the growth cone to bias the direction of axon extension. These recent advances allow us to propose a unifying model of how the growth cone translates shallow gradients of environmental information into polarized activity of the steering machinery for axon guidance.

Asymmetric clathrin-mediated endocytosis drives repulsive growth cone guidance.

Asymmetric Ca(2+) elevations across the axonal growth cone mediate its turning responses to attractive and repulsive guidance cues. Here we show that clathrin-mediated endocytosis acts downstream of Ca(2+) signals as driving machinery for growth cone turning. In dorsal root ganglion neurons, the formation of clathrin-coated pits is facilitated asymmetrically across the growth cone by a directionally applied chemorepellent, semaphorin 3A, or by Ca(2+) signals that mediate repulsive guidance. In contrast, coated pit formation remains symmetric in the presence of attractive Ca(2+) signals. Inhibition of clathrin-mediated endocytosis abolishes growth cone repulsion, but not attraction, induced by Ca(2+) or extracellular physiological cues. Furthermore, asymmetric perturbation of the balance of endocytosis and exocytosis in the growth cone is sufficient to initiate its turning toward the side with less endocytosis or more exocytosis. With our previous finding that growth cone attraction involves asymmetric exocytosis, we propose that the balance between membrane addition and removal dictates bidirectional axon guidance.

The nitric oxide-cGMP pathway controls the directional polarity of growth cone guidance via modulating cytosolic Ca2+ signals.

Asymmetric Ca(2+) signals across the growth cone mediate attractive or repulsive axon guidance depending on the occurrence of Ca(2+)-induced Ca(2+) release (CICR) through ryanodine receptors (RyRs). Although the neuronal isoform of nitric oxide (NO) synthase (nNOS) is highly expressed in developing dorsal root ganglion (DRG) neurons, the role of NO in axon guidance remains essentially unknown. Here we report that the NO-cGMP pathway negatively regulates CICR to control the directional polarity of DRG axon guidance. Intracellular levels of NO and cGMP depend on extracellular substrates: laminin activates the NO-cGMP pathway, whereas the adhesion molecule L1 does not. The activity of NO and cGMP determines the turning direction of growth cones with respect to asymmetric Ca(2+) signals that are produced by photolysing caged Ca(2+). The Ca(2+) signals cause growth cone repulsion on a laminin substrate, which is converted to attraction by pharmacological blockade of the NO-cGMP pathway or genetic deletion of nNOS. Conversely, Ca(2+)-induced growth cone attraction on an L1 substrate is converted to repulsion by increasing NO levels. Such NO-mediated switching of turning direction involves the regulation of CICR through RyRs. Furthermore, growth cone repulsion induced by an extracellular gradient of a physiological cue, neurotrophin-4, is dependent on Ca(2+) signals and converted to attraction by inhibiting the NO-cGMP pathway. These results suggest that, on contact with different adhesive environments, growth cones can change their turning responses to axon guidance cues by modulating CICR via endogenous NO and cGMP.

DSCAM deficiency causes loss of pre-inspiratory neuron synchroneity and perinatal death.

Down syndrome cell adhesion molecule (DSCAM) is a neural adhesion molecule that plays diverse roles in neural development. We disrupted the Dscam locus in mice and found that the null mutants (Dscam(-/-)) died within 24 h after birth. Whole-body plethysmography showed irregular respiration and lower ventilatory response to hypercapnia in the null mutants. Furthermore, a medulla-spinal cord preparation of Dscam(-/-) mice showed that the C4 ventral root activity, which drives diaphragm contraction for inspiration, had an irregular rhythm with frequent apneas. Optical imaging of the preparation using voltage-sensitive dye revealed that the pre-inspiratory neurons located in the rostral ventrolateral medulla and belonging to the rhythm generator for respiration, lost their synchroneity in Dscam(-/-) mice. Dscam(+/-) mice, which survived to adulthood without any overt abnormalities, also showed irregular respiration but milder than Dscam(-/-) mice. These results suggest that DSCAM plays a critical role in central respiratory regulation in a dosage-dependent manner.