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Boston University OpenBU http://open.bu.edu Theses & Dissertations Boston University Theses & Dissertations 2015 The role of the Golgi apparatus in neuronal polarity Ash, Tyler Dale http://hdl.handle.net/2144/16363 Boston University BOSTON UNIVERSITY GRADUATE SCHOOL OF ARTS AND SCIENCES Thesis THE ROLE OF THE GOLGI APPARATUS IN NEURONAL POLARITY by TYLER DALE ASH B.S., Michigan State University 2013 Submitted in partial fulfillment of the requirements for the degree of Master of Arts 2015 Approved by First Reader __________________________________________________ Angela Ho, Ph.D. Assistant Professor of Biology Second Reader _______________________________________________ Cynthia Bradham, Ph.D. Assistant Professor of Biology ACKNOWLEDGEMENT I would like to thank the many people who helped make this Master’s Thesis possible. I would like to thank everyone from the Ho lab for their help and support. I would like to specifically thank my advisor, Dr. Angela Ho, for all of her guidance and expertise. I would like to thank the entire faculty involved in my graduate education for continued support in and out of the classroom. I particularly want to thank Dr. Ulla Hansen for her counseling from start to finish as my program director, and Dr. Cynthia Bradham for her career counseling through graduate school. I am grateful for the support network provided by my fellow graduate students who have been friends, colleagues, and mentors. I want to thank Greg Dillon for teaching me everything I know in lab, Andressa Mota for going through it all with me, and Patrick Stoiber for being the JD to my Turk. I want to thank my family, old and new, who have always encouraged me to pursue my dreams, and tolerated my science rambles, as well as jokes. Most of all I want to thank my loving wife Erin for all her help and support. For all of the guidance you have given me. For always being there when I needed someone to talk to, someone to encourage me, and someone to support me. For providing me with the most conducive work environment through graduate school. I would not have made it this far without her. Thank you. iii THE ROLE OF THE GOLGI APPARATUS IN NEURONAL POLARITY TYLER DALE ASH ABSTRACT The Golgi apparatus has always been an interesting organelle of study because of its unique morphology as well as the critical roles it plays in cell biology. It is situated next to the endoplasmic reticulum and secreted proteins must pass through the Golgi vesicular pathway for modifications and targeting. In addition, the Golgi apparatus plays an essential role in establishing cellular polarity. Cell polarity refers to difference in orientation of cell structures spatially, and is involved in establishing functionality. The Golgi apparatus establishes cell polarity in various ways including orienting itself spatially, biasing vesicular trafficking within the cell, and most importantly through its role as a microtubule organizing center. The cytoskeleton provides the structural framework for cells. Microtubules nucleated from the Golgi-dependent microtubule organizing center result in an asymmetric cytoskeleton. An asymmetric cytoskeleton is essential to establishing cell polarity. Neurons require cell polarity to establish the essential structures such as the axon and dendrites. The Golgi apparatus establishes neuronal polarity through its extensive network of associated proteins. In this review, we will discuss the growing evidence supporting the role of the Golgi apparatus in establishing neuronal iv polarity. TABLE OF CONTENTS INTRODUCTION: ..................................................................................................1 AIM: .......................................................................................................................2 BACKGROUND INFORMATION: .........................................................................2 Golgi Apparatus ...............................................................................................2 Golgi Structure and Function ..........................................................................2 Microtubule Organizing Center .......................................................................7 Neurodevelopment ...........................................................................................8 Brain Cells ......................................................................................................8 The Basics of Brain Development ................................................................10 Cerebral Cortex ............................................................................................12 Polarity of Cells .............................................................................................13 Neuronal Migration .......................................................................................15 Secretory Pathway Involvement with Growing Dendrites and Axons ...........18 GOLGI APPARATUS ASSOCIATED PROTEINS: .............................................19 Rab GTPases ..................................................................................................19 Activation of Rab Proteins ............................................................................20 Rab Proteins in Neuronal Polarity ................................................................21 Cytoplasmic Linker Associated Proteins ....................................................22 Structure and Function of CLASPs ...............................................................23 v CLASP2 and Protein 4.1R ............................................................................26 CLASP2 and LL5β ........................................................................................26 CLASPs and the Golgi ..................................................................................27 CLASP2 Regulates Neuronal Polarity ..........................................................29 GRIP and Coiled-Coil Domain Containing Protein 185 ...............................30 Structure of Golgin Proteins .........................................................................31 GCC185 and Rab GTPase Binding Sites .....................................................34 GCC185 and AP-1 Protein ...........................................................................34 GCC185 and CLASPs ..................................................................................35 CONCLUSION: ...................................................................................................38 JOURNAL ABBREVIATIONS: ...........................................................................42 REFERENCES: ...................................................................................................43 CURRICULUM VITAE: ........................................................................................50 vi LIST OF FIGURES Figure 1: Two models for membrane traffic through the Golgi apparatus 6 Figure 2: Schematic diagram of radial-directed cortical neuronal migration 13 Figure 3: Diagram of layering in human cortical development stages 16 Figure 4: Cycling of Rab from active to inactive states 21 Figure 5: Representation of microtubule nucleation and catastrophe 24 Figure 6: CLASPs in the cytoplasm are involved in microtubule nucleation from the Golgi apparatus ultimately establishing cell polarity 28 Figure 7: Schematic representation of known human golgin domains 32 Figure 8: Schematic representation of GCC185 fragments 37 vii LIST OF ABBREVIATIONS Abbreviation +TIP AP1 aPKC ArfGEF2 Arl BIG2 CC CLASP EB1 FERM Fz GAP GCC185 GDP GEF GRASP GRIP GSK3β GTP PHLDB2 SR TMD TOG TOGL Vangl2 Full Text Plus-End Tracking Protein Adaptor Protein 1 Atypical PhosphoKinase C ADP-Ribosylation Factor Guanine Nucleotide Exchange Factor ADP Ribosylation factor-Like Brefeldin A-Inhibited Guanine Nucleotide Exchange Factor Coiled-Coil Cytoplasmic Linker Associated Protein End Binding 1 4.1 Ezrin, Radixin, Moesin Frizzled GTPase Activate Protein GRIP and Coiled-Coil Domain Containing 185 kiloDaltons Guanosine DiPhosphate Guanine Exchange Factor Golgi Reassembly and Stacking Proteins Golgin-97, Ranbp2α, Imh1p, and P230/golgin-245 Glycogen Synthase Kinase-3 Beta Gaunosine TriPhosphate Pleckstrin Homology-Like Domain B2 Serine Rich Transmembrane Domain Tumor Over Expressed Tumor Over Expressed Like Van Gogh Like 2 viii 1 INTRODUCTION: Research of the Golgi apparatus initially focused on the unique structure of the organelle and its role in protein trafficking. It is a dynamic organelle that receives vesicles containing proteins from the endoplasmic reticulum. As these vesicles traverse through the Golgi, resident proteins add post-translational modifications. The proteins are then trafficked to their destination. Proteins that pass through the Golgi are often targeted for secretion. Because of this, the Golgi apparatus is the center of the secretory pathway. Recent experiments aiming to elucidate the function the Golgi apparatus plays in cellular polarity have been performed in multiple model organisms ranging from Drosophila to primates. Polarity of cells provides functionality by orienting organelles spatially in a targeted direction. Cell polarity is required in most cell types. The Golgi apparatus and its network of associated proteins are required in establishing cell polarity. This occurs through Golgi-dependent nucleation of microtubules. This association with the cytoskeleton allows orientation of the Golgi during cell polarization. Neurons are a unique cell type that requires cell polarity to establish cellular structures essential for neurodevelopment. Neurons have two unique structures, axons and dendrites, which set them apart from other cell types. The axon is a long singular projection from the soma, or cell body, that are used to send information from the neuron. There are multiple dendrites that branch out 2 from the soma. Dendrites receive information from neurons. These unique structures are required for proper functioning of neurons, as well as for migration during neurodevelopment. Cell polarity is essential in establishing these dendritic and axonal projections in neurons. Interestingly, the Golgi apparatus has been shown to be involved in establishing polarity in various cell types including neurons. AIM: This review aims to detail the role of the Golgi apparatus in establishing neuronal polarity during neurodevelopment. BACKGROUND INFORMATION: Golgi Apparatus Golgi Structure and Function The Golgi apparatus is a ribbon-shaped organelle made up of disk-like structures called cisternae. The resident proteins of the cisternae are responsible for post-translational modifications such as adding oligosaccharide side chains. The Golgi apparatus is considered to be divided into three functional and spatial segments. Structurally, the cis face of the Golgi is the portion adjacent to the 3 endoplasmic reticulum. The middle portion of the Golgi is termed the medial Golgi. The trans-Golgi is the portion facing the plasma membrane. The transGolgi network is the final portion of the Golgi, which sends off vesicle buds targeting proteins to their final destination (Glick and Nakano, 2009). A crucial function of the Golgi apparatus is to transport cargo proteins in vesicles from the endoplasmic reticulum to target destinations within the cell or to the cell membrane for exocytosis, and thus is referred to as the secretory pathway. The directionality from the cis-Golgi face towards the trans-Golgi face is anterograde movement. Vesicles can also be transported from the trans face towards the cis face of the Golgi and continue on to the endoplasmic reticulum, which is called retrograde transport, and is usually involved during endocytosis from the cell membrane to the Golgi apparatus. Proteins discovered early in the history of Golgi studies appeared to exist to maintain the structural integrity of this organelle while vesicles were moving bidirectionally through the Golgi. There are two common morphologies of the Golgi apparatus, stacked or ribbon. A Golgi stack is a round or globular-like shape of the Golgi. A stacked Golgi is usually found in a symmetrical cell where transport vesicles leave the Golgi in all directions. This requires a more circular shape for the Golgi because it is continuously sending out and receiving vesicles to and from all directions of the cell. A Golgi ribbon is a long flat morphology of the organelle and is associated with an asymmetric cell growth or shape. If one 4 end or portion of a cell is growing or moving then the Golgi will orient itself towards that region while supplying it with vesicular bound proteins as well as lipids for the expanding membrane. Two Golgi Reassembly and Stacking Proteins (GRASP) known as GRASP55 and GRASP65 were shown to cross-link adjacent cisternae to support the stacked and ribbon structure of the organelle (Lowe, 2011). When both GRASP proteins were depleted, the Golgi structure is abolished resulting in fragmentation of the Golgi apparatus, while singular loss of a GRASP leads to a reduction in the number of cisternae (Xiang and Wang, 2010). Intra-Golgi cargo transport is accomplished through the use of coat proteins. COPII coated vesicles are involved in anterograde movement of Golgi cisternae, and COPI coated vesicles are involved in retrograde movement of Golgi cisternae. COPII and COPI coat the budding vesicles to provide structural support to the vesicle membrane and support fusion to Golgi cisternae using SNARE complexes (Szul and Sztul, 2011). There are two current theories regarding trafficking through the Golgi. The first is the stable compartments model (Figure 1a). In this model, the various regions of the Golgi (cis, medial, and trans-Golgi) are seen as distinct entities that maintain a specific set of resident proteins. Vesicles are donated from the endoplasmic reticulum to the Golgi through COPII vesicles and then COPI vesicles are responsible for coating vesicles in intra-Golgi transportation. The 5 stable compartments model implies that the Golgi is a relatively static organelle. The second theory that is most strongly supported by current literature is the cisternal maturation model (Figure 1b). This model postulates that the Golgi compartmental proteins common to the sections of the Golgi form de novo, describing the Golgi as a dynamic organelle. The Golgi proteins carried in the budding vesicles are kept in the proper region of the Golgi through signal sequences that target them to their region of function. Clathrin-coated vesicles bud from the trans-Golgi network towards their targeted destination. The cisternal maturation model is the currently supported model, and it accounts for the dynamic range of function by the Golgi apparatus in an ever-changing cell that has to constantly respond to inter- and intra-cellular changes (Glick and Nakano, 2009; Pelham, 2001). 6 Figure 1: Two models for membrane traffic through the Golgi apparatus. (a) Stable compartments model. (b) Cisternal maturation model (Glick and Nakano, 2009). Important findings regarding protein localization within the secretory pathway support the cisternal maturation model. As previously mentioned, there are sequences that target vesicular proteins to specific locations. One example is the C-terminal KDEL amino acid sequence in soluble endoplasmic reticulum proteins. This sequence is recognized by KDEL receptors in post-endoplasmic reticulum Golgi compartments and shuttles proteins containing the amino acid 7 sequence from the Golgi back to the endoplasmic reticulum (Munro and Pelham, 1987; Pelham et al., 1988). Another example includes recycling of glycosyltransferases within the Golgi (Storrie et al., 1998). The ability, provided by the KDEL sequence, to localize Golgi processing proteins is crucial for proper function of the secretory pathway. Without them, resident proteins would quickly become depleted after multiple vesicles are trafficked away, and Golgi regions would lose functional processing properties. Microtubule Organizing Center The role of the microtubule organizing center in animal cells is to act as the nucleation site for microtubules. Traditionally, centrioles are credited as the microtubule organizing center of a cell; however, non-traditional microtubule organizing centers may refer to other structures such as the pericentriolar matrix, or centrosomes (Kloc et al., 2014). Recent research identified the Golgi apparatus as a non-traditional microtubule organizing center that can nucleate microtubules with a slightly unique function (Zhu and Kaverina, 2013). Golgi-derived microtubule organizing centers are required for cell polarity. Centriole-based microtubule organizing centers usually provide a relatively symmetrical microtubule formation. Non-centrosomal microtubules derived from a Golgi-based microtubule organizing center have been found to be critical to vesicular trafficking of Golgi vesicles, as well as the directionality of cellular 8 processes, also known as cell polarity. Cell polarity signifies that a cell is asymmetrical, adopting an orientation in one direction for a specific function. Because of this, microtubules are crucial in the function of the Golgi. The plusend of a microtubule refers to the end that is polymerizing. Microtubule minusends interact with the Golgi apparatus along with the motor protein dynactin enabling the essential motor coordination of Golgi vesicles (Lowe, 2011; Vaughan, 2005). Neurodevelopment Brain Cells Cell polarity plays an essential role in development of neurons. The neuron is the most well-known cell type of the brain. A neuron is defined by its ability to send electrical action potentials through neuronal circuitry and form connections, referred to as synapses between two neurons. Synapses are the essential form of communication in the brain and occur through passage of electrochemical information from one neuron to another. The information can cause excitation or inhibition of other neurons and is passed through endogenous neurotransmitters, which are packaged in vesicles in the cell and are released through exocytosis of the vesicle. There are numerous neurotransmitters that are usually categorized as peptides, amino acids, or 9 monoamines. Neurons have dendrites and axons, which are uniquely extensive for varying neuronal cell types. Generally, a neuron has one axon that sends signals to other neurons. There are multiple dendritic branches from a single neuron and they receive signals sent by axons. The dendritic branches allow one neuron to simultaneously receive input from multiple neurons (Stiles and Jernigan, 2010; Tau and Peterson, 2009). Maturation of white matter and gray matter is essential during proper development of the brain. Gray matter contains nerve cell bodies and branching dendrites, and is where the main functions of the brain occur, such as the relay of somatosensory information. White matter consists of nerve fibers as well as myelin sheaths and glial cells. The white matter connects different functional regions of the brain together forming complex networks and is the matter that makes up the corpus callosum (Dubois et al., 2014). Glia is another major cell type in the brain. As brain size increases, the proportion of glia to neurons increase. There are only a few glia in nematode brains, whereas up to 90% of the human brain consists of glia. Glia were originally thought to only have a passive role in supporting neurons by providing a suitable environment. Glia have since been found to have an important function in assisting informational processing in adulthood, as well as in development (Allen and Barres, 2009; Barres and Barde, 2000). 10 There are three types of glia: oligodendrocytes, astrocytes, and microglia. Oligodendrocytes wrap axons in myelin to facilitate faster transfer of action potential along the axon. Astrocytes provide neurons with a homeostatic environment by providing energy and substrates for neurotransmitters. Microglia act as specialized immune cells, which monitor the brain for damage or infection by engulfing neuronal debris and dead cells. In addition, microglia are involved in synaptic remodeling in development by pruning unnecessary synaptic connections (Allen and Barres, 2009; Barres and Barde, 2000). The Basics of Brain Development Neurodevelopment is very complex and has drawn a lot of interest in its study because of the drastic phenotypic effects that can occur from small changes in basic cell function. There is a large body of work covering many levels of neuronal development ranging from the microscopic level to whole systems analysis (Stiles and Jernigan, 2010; Tau and Peterson, 2009). Research has advanced markedly in the field of neurodevelopment within the last ten years; however, with each new insight comes more potential questions, making the study of neuronal development as important today as it was 100 years ago. Development of the human brain spans decades to produce a fully matured brain. In humans, initial development begins in gestational week three with differentiation of neural progenitor cells, and requires specific environmental 11 and genetic cues to occur properly. The first step is production of neuronal stem cells, or neural progenitor cells. Through a complex cascade of genetic signaling molecules, neuronal progenitor cells populate the region called the neural plate. During neurulation, which is the development of the nervous system, the neural plate compresses towards the notochord forming the neural tube, the precursor to the spinal cord. Transcription factors such as bone morphogenetic proteins and sonic hedgehog are expressed in gradients along the neural tube and lead to specific activation of developmental genes (Barres and Barde, 2000; Stiles and Jernigan, 2010; Tau and Peterson, 2009). Synaptogenesis, the development of synapses, is a dynamic process, which peaks in humans during postnatal development, and results in a high density of neuronal synapses. As was mentioned previously, a synapse is the connection between two neurons where synaptic information is passed. As synapses are formed, they are concurrently pruned through phagocytosis, resulting in clearance and necrosis of up to 50% of synapses and neurons, respectively (Purves and Lichtman, 1980; Rakic et al., 1986; Stiles and Jernigan, 2010; Tang et al., 2014). Despite this drastic reduction in neuronal population; intellectual capabilities are not compromised, as intelligence does not correlate with brain weight. The significance of the neuronal circuits, connections, and quality of synapses is important to intellectual processing and defects in proper pruning of synapses have been implicated in frontal and parietal lobes of autism 12 spectrum disorder patients (Bourgeron, 2009; DeFelipe, 2011; Peça and Feng, 2012). Cerebral Cortex The cerebral cortex is the outer layer of the cerebrum and is composed of folds and indendations of gray matter making the gyrus and sulcus, respectively. The extensive folding provides an increase in surface area while maximizing the available volume of space the brain can occupy. The cortex develops in an inside out manner. During development, the progenitor cells divide symmetrically until there is a substantial population of progenitors. Once this occurs, progenitor cells begin asymmetric cell division producing one renewed progenitor cell and one dissimilar daughter cell. In the cortex, the primary progenitor cell is a radial glial cell that undergoes asymmetric cell division to produce another radial glial cell and a neuron (Miyata et al., 2001; Noctor et al., 2002). The neuron then migrates from the ventricle towards the pia (radially) along the radial glial projection. Subsequent divisions of the radial glial cell result in the younger neuron migrating radially past the older neuron to the outer layer (Figure 2) (Bielas et al., 2004). Thus functional migration is essential for proper neurodevelopment. 13 Figure 2: Schematic diagram of radial-directed cortical neuronal migration. Neurons (green) are generated in the ventricular zone and migrate along radial glial cells (orange) through the subventricular zone, intermediate zone, subplate, and cortical plate. The cortical plate is established in an inside-out pattern so that later-generated neurons (progressively darker green shading) destined for more superficial layers bypass earlier-generated neurons in deeper layers. The marginal zone includes horizontally oriented Cajal-Retzius cells (red) (Bielas et al., 2004). Polarity of Cells During neurodevelopment, it is important that cells undergo asymmetric cell division to continue the progenitor cell line and produce neurons. A crucial component of asymmetric cell division is planar cell polarity. Planar polarity is described as a uniform orientation of a population of cells within a 2-dimensional 14 space. Planar cell polarity is a common property in epithelial tissue. In the animal kingdom, planar cell polarity has been widely studied in Drosophila, and the principles of planar cell polarity in asymmetric cell division are conserved in mammalian cells (Goodrich and Strutt, 2011; Guilgur et al., 2012). Core proteins of the planar cell polarity pathway have been shown to be expressed asymmetrically in the cell including the transmembrane receptors Frizzled (Fz) and Van Gogh Like 2 (Vangl2) (Goodrich and Strutt, 2011). These two transmembrane receptors are implicated as core components of the Wnt7a pathway which is known to promote the planar cell polarity pathway (Wang and Nathans, 2007). Using 3D reconstruction of the mitotic spindle, a study in mice described asymmetric cell division dependence on spindle size asymmetry during mammalian corticogenesis (Delaunay et al., 2014). Mitotic spindle size asymmetry is a measure of the volume of microtubule spindles during metaphase. Studies have found that Vangl2 and the Wnt7a pathway control the magnitude of spindle size asymmetry. During asymmetric cell division of a radial glial cell, the larger spindle volume leads to a neuronal cell, and smaller volume portion of the cell resulted in a radial glial cell. In recent studies spindle size asymmetry was shown to occur during corticogenesis in the macaque monkey (Delaunay et al., 2015). Taken together, this suggests that cell polarity is essential in neurodevelopment. 15 Neuronal Migration As previously mentioned, once the neuron is produced from asymmetric cell division of a radial glial cell, it must migrate radially to produce proper layering of the brain. In Figure 2, the cell architecture/structure of the brain can be partitioned into six layers within the cortical plate (Figure 3). Neurons migrate along the radial glial cell until they receive a stop signal. In Figure 3A, the CajalRetzius cells are positioned in yellow just before the pia matter at the extreme outer boundary of the brain. Cajal-Retzius cells produce the molecular stop signal, Reelin, a protein recognized by the neurons signaling an end of their migration (Bielle et al., 2005; Huang, 2009). Migration defects resulting from dysfunctional Reelin lead to severe brain malformation such as inversion of the cortical layers. Complete loss of Reelin can result in a form of lissencephaly (smooth brain). Each layer has its own unique density of neurons, number of synapses, length and branching of dendrites and/or axons, as well as inputs and outputs of information (Bielas et al., 2004; Stiles and Jernigan, 2010; Tau and Peterson, 2009). Neuronal migration requires a complex coordination in movement of organelles between the centrioles, the Golgi, and the nucleus. These organelles are implicated in associations with microtubule and axonal nucleation along with the motor protein dynactin. Not all neurons migrate in the same fashion. Some 16 produce continuously changing filopodial projections; however, they generally have a long leading process that resembles a growth cone with dynamic filopodia. Despite the subtle differences in migration of neurons, it is clearly established that neuronal migration requires an intense and dynamic reconstruction of the cytoskeleton in close association with known microtubule organizing centers (Cooper, 2013). Figure 3: Diagram of layering through stages of human cortical development stages. A) Migration of neurons in an inside out pattern with the darker blue/purple being the older neurons. B) Layering changes through development from left to right, producing the final layering of the cortical plate shown on the right (Stiles and Jernigan, 2010). 17 Secretory trafficking regulates growth of the plasma membrane in cell motility (Etienne-Manneville and Hall, 2003). Mutations that disrupt functional cytoskeletal reorganization and their interaction with associated proteins have profound effects on neuronal migration. Mutations in filamin disrupt actin-binding proteins leading to the human disease periventricular nodular heterotopia, which is a human disease caused by improper migration of neurons in utero. Individuals with periventricular nodular heterotopia will usually experience mild seizures in their teen years and have normal intelligence; however, will rarely have learning disabilities. Disruption of vesicle trafficking within the Golgi also causes periventricular nodular heterotopia, epilepsy, as well as microcephaly (small brain size) from mutations in the gene ADP-ribosylation factor guanine nucleotide-exchange factor 2 (ArfGEF2) which codes for the protein brefeldin A-inhibited GEF2 (BIG2). BIG2 has functional importance in vesicular trafficking from the trans-Golgi network. Double mutations of ArfGEF2 and/or disruption of BIG2 activity resulted in loss of vesicular trafficking-dependent proliferation and migration of neurons during development (Sheen et al., 2004). It is clear from previous research that defects in proteins implicated in the Golgi vesicular trafficking network such as Reelin, filamin, and BIG2 have significant effects on neuronal migration (Bielas et al., 2004). 18 Secretory Pathway Involvement with Growing Dendrites and Axons Functional neurons must establish an axon as well as branching dendrites. The dendritic arborization and the aspects of axonal growth have significance for the establishment of neuronal circuits (Tau and Peterson, 2009). To produce dendrites and axons, the membrane of the cell must expand greatly to compensate for the extending filopodia. This is made possible through the secretory pathway, which is the largest source of additions to the plasma membrane through vesicular trafficking (van Vliet et al., 2003). The Golgi apparatus is critical in the extension of dendrites. In the soma, a polarized Golgi apparatus orients towards the growing dendrite and is required for polarized dendritic arbors. Small Golgi outposts were also found at branch points of growing dendrites, but are excluded from axons. Overexpression of GRASP65 leads to dispersal of Golgi vesicles. Golgi dispersal depletes Golgi polarization towards the longest dendrite. This results in loss of dendritic polarity, causing symmetric dendritic arbors, and the longest axon was not formed (Horton et al., 2005). Unlike dendrites, axonal growth is not regulated through control of the secretory pathway. During development, the neuron receives signals from amacrine cells, unique unipolar neurons, to irreversibly switch from axonal growth to dendritic, suggesting separate modes of outgrowth. More recent work identified that when expression of Sar1, a protein involved in vesicle budding 19 from the endoplasmic reticulum to the Golgi, was knocked down using siRNA, a significant loss of dendritic growth was observed with no noticeable affect to axon length (Goldberg et al., 2002). This implicates Golgi involvement in dendritic growth and that axonal growth relies on different cellular and molecular mechanisms which may help to establish their unique morphologies. GOLGI APPARATUS ASSOCIATED PROTEINS: Given the integral role the Golgi apparatus plays in the secretory pathway, neuronal migration, and a plethora of other physiological cell processes, it is no surprise there are numerous Golgi associated proteins. This chapter focuses on proteins that are associated with the Golgi and its role in neuronal polarity. As previously discussed, there are proteins involved in processing proteins and lipids as they pass through the secretory pathway. These proteins will not be discussed in this chapter unless they have a specific role in the Golgi apparatus relating to neuronal polarity. Rab GTPases Rab proteins are notably associated with the Golgi apparatus. Rab proteins are in the Ras superfamily of small GTPases. This family is categorized by the function of the small GTPase, meaning it has a catalytic site that can bind 20 and hydrolyze guanosine triphosphate (GTP). Rab proteins are highly conserved in eukaryotes, and there are more than 50 Rab genes in the human genome alone. Rabs are transmembrane proteins localized to the cytosolic face of organelles by Rab escort proteins, and anchored via a prenyl group on the Cterminus. In relation to the Golgi, Rab proteins are involved in trafficking through the secretory pathway. Once bound by an effector protein, Rab modulates vesicle formation, membrane fusion, and membrane trafficking. Multiple Rab proteins are essential in the functions performed by the Golgi apparatus (Liu and Storrie, 2012, 2015; Stenmark and Olkkonen, 2001). Activation of Rab Proteins The activity of Rab proteins, like other Ras superfamily small GTPases, depends on their binding of GTP or GDP (Figure 4). For example, Rab bound by GTP will be termed as active Rab. Inactive GDP-Rab is activated by a guanine exchange factor (GEF), which switches a GDP for GTP. GTP-Rab is active and can act on its target proteins to influence vesicular trafficking and fusion. To deactivate Rab, the small GTPase domain of Rab must hydrolyze the γphosphate of the GTP; however, the domain cannot initiate hydrolysis alone. Rab’s GTPase activity will be activated by a GTPase activating protein (GAP), which catalyzes cleavage of the tertiary phosphate by the small-GTPase site 21 resulting in GDP-Rab. Inactive Rab can be shuttled by Rab escort protein, or reactivated by a GEF (Stenmark and Olkkonen, 2001). Figure 4: Cycling of Rab GTPases from active to inactive states. Active GTP-Rab is inactivated through GAP catalyzing the Rab small GTPase resulting in cleavage of the γ-phosphate leaving GDP-Rab. GDP-Rab can be translocated by REP. GDP-Rab is activated by a GEF switching a GTP for the GDP resulting in active GTP-Rab (Stenmark and Olkkonen, 2001). Rab Proteins in Neuronal Polarity A study in 2004 implicated a mutation of the gene ADP ribosylation factor guanine nucleotide-exchange factor2 (ArfGEF2) as a cause of the neurological disorder periventricular nodular heterotopia, which is a result of improper neuron migration (Bielas et al., 2004; Sheen et al., 2004). ArfGEF2 is a GEF for Rab proteins. ArfGEF2 is known to localize to the Golgi and be involved in membrane trafficking. Because of this, a group performed studies of ArfGEF2 involvement in neuronal migration. They found that the Rab GTPase-dependent pathways have 22 roles in neuronal maturation and migration, and inhibition of Rab5 and Rab11 disrupts neuronal migration. This supports the model that Rab5 dependent endocytosis of membranes is involved in recycling N-cadherin from the trailing process to the leading edge. N-cadherins are transmembrane proteins required for cell adhesion during migration. Rab dependent recycling of N-cadherin from the trailing process to the leading edge is required to promote continued leading edge adhesion during neuronal migration (Kawauchi et al., 2010). The Golgi-associated Rab proteins are known to be integral to Golgi function, specifically in the Golgi’s ability to organize membrane trafficking (Liu and Storrie, 2012, 2015). Rab5 has been shown to be integral to Golgi trafficking of multiple proteins, including those important to neuronal migration (Fukuda et al., 2013). This information shows that certain Rab proteins are involved in establishing neuronal polarity through their interactions with the Golgi apparatus. Cytoplasmic Linker Associated Proteins Cytoplasmic linker associated proteins (CLASPs) are microtubule linking proteins in a family of plus-end tracking proteins (+TIPs). CLASPs are highly conserved across animal, plant, and fungal kingdoms (Galjart, 2005). They are known to be involved in attaching microtubules to the cell cortex. CLASPs direct microtubule dynamics, facilitate the relay of information within the cytoskeleton, and modulate the orientation of growth. Because of the range of functions 23 CLASPs are involved in, they are essential for proper growth in developing neurons (Akhmanova et al., 2001). There are two mammalian CLASP homologs known as CLASP1, which is ubiquitous, as well as CLASP2, which is brain specific. CLASPs are integral in establishing cell polarization and migration (Beffert et al., 2012; Drabek et al., 2006; Efimov et al., 2007; Stramer et al., 2010). Structure and Function of CLASPs The function of CLASP proteins is elucidated by their structure. The common domains of CLASP proteins are tumor over expressed (TOG)-like (TOGL) domains, as well as serine, proline, and arginine rich domains (SR-rich domains). In human CLASP proteins, there are three TOGL domains that stretch from the N-terminus to the middle of the protein. TOGL 1-3 are required for CLASP binding to microtubules. Each TOGL domain has differing affinity for microtubules. This is thought to provide CLASP proteins with varying levels of microtubule binding capabilities to increase versatility of CLASPs in vivo (Akhmanova et al., 2005; Al-Bassam and Chang, 2011). Structural analysis of TOGL domains showed they are not responsible for direct binding of CLASPs to microtubules. Instead, C-terminal to TOGL domains are SR-rich domains that have stretches of positively charged amino acids. These stretches are relatively unstructured in their exact sequence order; 24 however, they are highly conserved across species. It is thought that the combined function of TOGL and SR-rich domains allows CLASP to act as a rescuing protein during microtubule catastrophe, an event in which microtubule bundles undergo an explosive shrinkage through filaments peeling off of the microtubule bundle (Figure 5) (Akhmanova et al., 2005; Al-Bassam and Chang, 2011). Figure 5: Representation of microtubule nucleation and catastrophe. a) GTP bound β-tubulin is represented in green, and α-tubulin is light blue and they form a straight dimer. After hydrolysis of the γ-phosphate GDP bound β-tubulin 25 undergoes a conformational change resulting a bend in the dimer. b) During microtubule assembly GTP tubulin is added at the plus-end of the microtubule. If the tubulin at the plus-end is dephosphorylated the dimers bend resulting in microtubule catastrophe. CLASPs binding to microtubules through TOGL domains provide structural support that can rescue microtubule catastrophe (AlBassam and Chang, 2011). Beyond interacting with microtubule networks directly, there are structural components of CLASPs C-terminal region that localize it in the cell. The S-X-I-P motif is an end binding 1 (EB1) interaction domain that allows EB1 and CLIP-170 binding to CLASP. EB1 proteins are conserved targeting proteins. CLIP-170 is a microtubule plus-end tracking protein known to modulate dynactin localization, and microtubule organization and linkage to endosomes. EB1 and CLIP-170 both target CLASP to the plus end of growing microtubules. CLASP proteins also have phosphorylation sites in the C-terminus that affect its localization. Glycogen synthase kinase-3 beta (GSK3β) is a serine-threonine kinase known to affect localization of CLASP to plus-ends through phosphorylation of CLASPs Cterminus. GSK3β acts as a guide through its regulation of CLASPs for microtubules dynamics in the sorting process from the trans-Golgi network (Akhmanova et al., 2005; Al-Bassam and Chang, 2011). CLASPs have various essential functions in the cell. They are required at the growing plus-end of microtubules, as well as for anchoring at the Golgi associated minus-end. CLASPs binding along their length also stabilize microtubules. These functions are made possible through CLASPs association 26 with various proteins. We will now discuss CLASPs associating proteins relevant to the Golgi apparatus, neurodevelopment, and neuron function in detail. CLASP2 and Protein 4.1R The protein 4.1R was recently found to be involved in localization of the brain specific CLASP2. Protein 4.1R is part of the large band 4.1 superfamily, defined by their highly conserved 4.1 ezrin, radixin, moesin (FERM) domain which functions to bind proteins and lipids (Chishti et al., 1998). Initially, 4.1R was found to regulate architecture of microtubules, centrosomes, endosomes, and the nucleus. Later work found that CLASP2 associates with protein 4.1R independently of microtubules. 4.1R modulates binding of CLASP2 to the plusends of microtubules by reducing the ability of GSK3β to phosphorylate the Cterminal region of CLASP2. Through this mechanism, 4.1R with CLASP2 exhibits control over microtubule dynamics and organization in the leading edge required for proper cell polarity that is essential for migration (Ruiz-Saenz et al., 2013). CLASP2 and LL5β The protein LL5β is a binding protein of phosphatidylinositol-3,4,5triphosphate. It is expressed by the gene pleckstrin homology-like domain B2 (PHLDB2) and is involved in intracellular signaling. LL5β is localized to the postsynaptic membrane because of its pleckstrin homology domain, where it 27 binds CLASP2 bound microtubules. Acetylcholine is an essential neurotransmitter in both the peripheral and central nervous system and is essential to the somatic nervous system. The acetylcholine receptor accumulates in neurons by anchoring to the cytoskeleton. LL5β, located at postsynaptic membranes, binds CLASP2, which captures microtubules that have anchored acetylcholine receptors. This focuses accumulation of acetylcholine receptors at postsynaptic membranes. This localization of acetylcholine receptors is required to capture acetylcholine signaling between neurons, and is organized by the LL5β and CLASP2 transport system (Basu et al., 2015). The association of LL5β with CLASP2 anchors the plus-end of microtubules to the plasma membrane, facilitating the establishment of neuronal polarity. CLASPs and the Golgi The functionality of the Golgi apparatus is dependent on CLASPs. Early on, CLASPs were observed to localize to the Golgi apparatus (Akhmanova etal., 2001). Later work in human cells by Efimov et al., (2007) established that CLASPs association is required for microtubule nucleation at the Golgi apparatus in a centrosome-independent manner. Microtubule structures were ablated using nocodazole, which results in depolymerization of cellular microtubules. Microtubule polymerization was then observed forming a centrosomal-dependent 28 network as well as non-centrosomal microtubule network nucleating at the Golgi apparatus (Efimov et al., 2007). Nucleation of microtubules at the Golgi apparatus microtubule organizing center requires gamma-tubulin to initiate polymerization, similar to centrosomal nucleation (Figure 6). CLASPs are recruited to the trans-Golgi network by the Golgi associated protein, GRIP and coiled-coiled domain containing 185 kilodaltons (GCC185). CLASPs bind to gamma-tubulin, which acts as a seed to begin microtubule polymerization. Not only are CLASPs bound at the minus-end (trans-Golgi network), but they are also required at the plus-end (the growing microtubule tip) for proper anchored growth of microtubules. If CLASP is only at the plus-end then treadmilling of microtubules occurs. However, if there is no CLASP present to anchor microtubules, then there is no microtubule growth originating from the Golgi apparatus (Efimov et al., 2007). Figure 6: CLASPs in the cytoplasm are involved in microtubule nucleation from the Golgi apparatus ultimately establishing cell polarity. M) The role of CLASPs in Golgi-dependent microtubule nucleation shows that GCC185 attracts CLASPs to the Golgi, which establishes γ-tubulin to initiate microtubule initiation. N) Golgidependent microtubule nucleation is essential in establishing cell polarity, which is involved in vesicular trafficking, migration, actin assembly (Efimov et al., 2007). 29 Interestingly, it was found that microtubules nucleated from the trans-Golgi network are preferentially oriented towards the leading edge of migrating cells. Centrosomal microtubule networks are symmetrical. This implies a specific mechanism of Golgi-derived asymmetric microtubule formation in polarized cells. These findings support the idea that the Golgi apparatus is a crucial microtubule organizing center for cell polarity and migration, and that CLASPs are required for this function (Efimov et al., 2007). CLASP2 Regulates Neuronal Polarity Brain specific CLASP2 is involved in cytoskeletal rearrangements required for neuronal polarity when forming dendritic and axonal projections. As was previously discussed, axonal and dendritic growth occur through two different mechanisms; however, both require dynamic rearrangement of the cytoskeleton to establish the drastic polarization of neurons (Craig and Banker, 1994). Until recently, it was unclear how microtubule rearrangements occurred during neuronal development of synapses. Expression of CLASP2 is enriched in neuronal growth cones and increases throughout neuronal development. When CLASP2 expression is knocked down, neurons developed stunted dendritic branching as well as shorter axons. In the presence of abundant CLASP2, neurons develop multiple axons 30 and have increased arborization of dendrites. It also resulted in a condensed Golgi, which is expected given the extensive relationship between CLASP2 and the Golgi apparatus. The overexpression of CLASP2 also resulted in an increase in synaptic vesicle release, a component of vesicular trafficking. This lines up with previous knowledge of CLASP2 involvement with the Golgi apparatus. CLASP2 directs neuronal polarity through its association with microtubule dynamics (Beffert et al., 2012). The microtubule plus-end tracking protein CLASPs are crucial in neuronal polarity. CLASPs are known to be highly conserved across species, indicating that they perform an essential function, and are known to have an extensive association with the Golgi apparatus and Golgi associated proteins. There are two mammalian homologs, CLASP1 and CLASP2, both of which are known to be integral in establishing polarity. CLASP2 is exclusively expressed in the brain, and aberrant regulation of CLASP2 results in faulty dendritic arborization and axonal projection of neurons. Taken together, this signifies that CLASP2 is an essential protein in establishing Golgi directed neuronal polarity. GRIP and Coiled-Coil Domain Containing Protein 185 GRIP and coiled-coil domain containing protein 185 kilo-daltons (GCC185) is a golgin protein. Golgins are a family of proteins containing coiled-coil domains that are peripherally associated with the Golgi apparatus and involved in 31 tethering events during fusion of membranes. They also provide organizational and structural support to Golgi cisternae. Depletion of golgins leads to a loss of the normal Golgi ribbon structure resulting in fragmented stacks of Golgi (Derby et al., 2007; Munro, 2011; Yoshino et al., 2003, 2005). Golgi vesicular trafficking is reliant on the trans-Golgi golgin, GCC185 for structural support, organization, membrane fusion, and protein recruitment. GCC185 has an intricate relationship in Golgi processes (Brown et al., 2011; Derby et al., 2007; Efimov et al., 2007; Hayes et al., 2009; Lin et al., 2011; Matsui et al., 2015). Because of this, GCC185 is essential in establishing Golgi-dependent cell polarity. Structure of Golgin Proteins Golgins are ubiquitously expressed within the cell and are conserved across multiple species. They have been described as tentacle-like projections from the Golgi apparatus providing tethering sites for other binding partners associated with the Golgi. The golgin family of proteins is grouped on the basis that they are bound to the Golgi periphery and share two common structural domains. A schematic representation of known golgins can be seen in Figure 7. The first requirement is that golgins have a C-terminal domain that attaches them to the Golgi apparatus, which varies depending on if the golgin is associated with cis-Golgi, medial-Golgi, or trans-Golgi cisteranae (Munro, 2011). 32 Figure 7: Schematic representation of known human golgin domains. Each line is a schematic of a human golgin protein with domains shown in different colors. The domains are represented as the approximate proportion of the protein based on structural analysis. Gray: predicted coiled-coil domains, yellow: GRIP domain, purple: GRAB domain, green: ALPS domain, red: transmembrane domain (TMD) (Munro, 2011). Golgins also have a coiled-coil domain that is structurally comprised of alpha-helices coiled together. This results in a rope-like structure that comprises the majority of golgin proteins. The coiled-coil domains are not generally conserved between golgins, and much of the space within the domain is unstructured leading researchers to believe these stretches of the domain allow golgins increased flexibility through hinge or extension regions (Munro, 2011). 33 Golgins that are localized to the trans-Golgi have a C-terminal (golgin-97, RanBP2α, Imh1p, and p230/golgin-245) GRIP domain. GCC2, the human homolog of GCC185, is a GRIP domain containing trans-Golgi golgin (Figure 7). The GRIP domain is comprised of an approximately 80 amino acid conserved motif that localizes GCC185 to the trans-Golgi. There has been much debate on whether or not the GRIP domain is localized to the Golgi through association with the small G protein ADP-ribosylation factor-like 1 (Arl1), with research supporting both sides. Some GRIP domain proteins have been shown to be involved with cargo export from the trans-Golgi network (Munro, 2011). Cis-Golgi golgins do not have one universal method of attaching to the Golgi apparatus. GM130 has a domain on the carboxy terminus that binds to the PDZ domain, a common attachment domain found in proteins involved in signaling pathways, of the Golgi structural protein GRASP65. Another cis-Golgi golgin, GMAP-210 utilizes a GRAB domain to attach to the Golgi, which functions similar to the GRIP domain discussed previously. The golgins that attach to the cytoplasmic surface of medial cisternae do so through anchoring into the membrane via their transmembrane domain (Munro, 2011). Establishment of neuronal polarity requires the Golgi apparatus during axonal growth and dendritic arborization. Golgins play an essential role in the Golgi apparatus by acting as tethering sites for Golgi associated proteins. The trans-Golgi golgin GCC185 is required for the association of multiple proteins 34 with the Golgi apparatus, including CLASPs. We will discuss the association between GCC185 and relevant Golgi associated proteins, as well as its role in establishing neuronal polarity. GCC185 and Rab GTPase Binding Sites Vesicular tethering at the Golgi apparatus is crucial for trafficking vesicles, and processing secreted and membrane bound proteins. The structure and function of the Golgi apparatus is sensitive to the loss of golgin proteins because of the role they play in tethering cisternae. Cells depleted of GCC185 have an accumulation of vesicles in the cytoplasm because they lose the ability to fuse to the trans-Golgi network (Reddy et al., 2006). GCC185 has binding sites for up to 14 Rab GTPases, which act in affiliation with Arl1 GTPases to localize GCC185 to the trans-Golgi network. Through Rab binding GCC185 is able to maintain normal Golgi structure and stack morphology, and functional loss of GCC185 results in fragmentation of the Golgi (Hayes et al., 2009). This establishes the model that GCC185 is a vesicular tether at the trans-Golgi network. GCC185 and AP-1 Protein Mannose 6-phosphate receptors are transmembrane glycoproteins that bind newly synthesized acid hydrolases in the trans-Golgi network, and participate in their delivery to lysosomes Ghosh et al., 2003). After mannose 6- 35 phosphate receptors deliver the cargo to the lysosomes they need to be recycled back to the trans-Golgi network. Cells depleted of GCC185 accumulate mannose 6-phosphate receptors in transport vesicles (Reddy et al., 2006). Clathrin adaptor proteins bind cytoplasmic domains of cargo proteins and recruit clathrin coating to induce vesicle budding. Clathrin adaptor protein 1 (AP-1) binds the cytoplasmic domain of mannose 6-phosphate receptors. Retrograde transport of mannose 6-phosphate receptors occurs by attachment of AP-1, which has a binding site on GCC185. The golgin protein, through its GRIP localization domain, shuttles the complex to the trans-Golgi network ultimately recycling the mannose 6-phosphate receptors (Brown et al., 2011). The AP-1 mediated vesicle tethering by GCC185 shows that the golgin plays an essential role in vesicular trafficking. As discussed previously, the Golgi apparatus needs to orient vesicular trafficking to establish neuronal polarity. Taken together, this indicates the GCC185 function in vesicular trafficking, plays a role in Golgi-dependent establishment of neuronal polarity. GCC185 and CLASPs Up to this point, we have discussed how GCC185 can be involved in vesicular trafficking and structural organization of the Golgi apparatus through association with other proteins. In regards to the role the Golgi apparatus plays in microtubule organization of cell polarity, the most important proteins GCC185 36 interacts with are CLASPs. As was previously established, CLASPs are required for Golgi structure and organization, and ultimately for establishing neuronal polarity. In this section, we will discuss how GCC185 and CLASP interaction is required for normal Golgi function and establishing cellular polarity through its control over microtubule dynamics. GCC185 is involved in recruiting the plus-end microtubule binding CLASPs to the trans-Golgi network in a similar fashion to how GCC185 tethers AP-1 to recycle mannose 6-phosphate receptor vesicles to the Golgi (Efimov et al., 2007; Hayes et al., 2009). This interaction acts to bridge together the transGolgi network with CLASP, which organizes Golgi-derived microtubules to maintain structural support and organization, and establish cell polarization and motility (Efimov et al., 2007; Miller et al., 2009). ADP-ribosylation factor-like 4A (ARL4A) is a GTPase that acts as a positive modulator of the GCC185-CLASP interaction. ARL4A interacts with the coiled-coil 2 (CC2) sub-region of GCC185 (Figure 8), in a GTP-dependent manner. Depletion of ARL4A or deletion of the ARL4A interacting region CC2 on GCC185 abolishes the interaction between GCC185 and CLASP resulting in fragmentation of the Golgi. Taken together, this data suggests that binding by ARL4A to GCC185 facilitates the interaction between GCC185 and CLASPs (Lin et al., 2011). 37 Figure 8. Schematic representation of GCC185 fragments. GCC185 is a representation of the full length protein. CC1, 2, and 3 are predicted CC domains. Full length GCC185 was fragmented into the various CC domains to determine which domains are required for protein-protein interactions. CC: coiled-coil, CTD: C-terminal domain, C270: C-terminal 270 amino acid fragment (Lin et al., 2011). The cell polarity protein complex comprised of PAR3, PAR6, and atypical phosphokinase-C (aPKC) is essential in several aspects of cell polarity. PAR6 acts as an adaptor protein holding aPKC and PAR3 together. PAR3 interacts with the brain specific CLASP2, and negatively regulates its interaction with GCC185 in epithelial cells. PAR3 complexes with CLASP2 by binding to the middle of the protein in the serine-rich region, and escorts aPKC to phosphorylate the C-terminal region of CLASP2 between amino acids 900-1000. Knockdown of either PAR3 or aPKC resulted in hyper accumulation of CLASP2 to the trans-Golgi network. This means that phosphorylation of CLASP2 by aPKC 38 inhibits the binding of GCC185 and CLASP2. The aberrant accumulation of CLASP2 at the trans-Golgi network resulted in fragmentation of the Golgi apparatus. PAR3 and aPKC regulation of CLASP2-GCC185 interaction controls Golgi organization, and ultimately cellular polarity (Matsui et al., 2015). Taken together this data suggests that PAR3 escort’s aPKC, via the PAR3, PAR6, aPKC complex, to CLASP2 where aPKC phosphorylates CLASP2s C-terminal region. Once phosphorylated CLASP2 is targeted to the plus-end of microtubules. This data concurs with results discussed previously that found GSK3β phosphorylation of the C-terminal region of CLASPs localize them to the plus-end of the microtubule structure. Loss of aPKC or PAR3 activity results in CLASP2 accumulation at the trans-Golgi network. These data suggest that the cell polarity complex regulates the interaction between GCC185 and brain specific CLASP2, modulating Golgi-dependent cellular polarity. CONCLUSION: The Golgi apparatus plays an essential role in establishing neuronal polarity through its function as a microtubule organizing center, and control of the secretory pathway. During development, the cortex requires proper migration of neurons to establish layering. To migrate, neurons send out a leading process that adheres to the extracellular matrix through binding of N-cadherins. This 39 provides leverage for locomotion of the cell. The Golgi apparatus is required for recycling of N-cadherin from the trailing edge to the leading edge. The Golgi apparatus also orchestrates trafficking of proteins and lipids towards the leading edge by establishing cell polarity. This occurs on two levels. First, the Golgi orients itself from the endoplasmic reticulum toward the leading edge. This results in a bias in vesicular trafficking towards the edge, increasing the total membrane in that region for expansion of filopodial projections, as well as proteins required for cell movement and adhesion. The second, and likely most important method of establishing cell polarity is through establishing a non-centrosomal microtubule organizing center. The Golgi apparatus establishes the microtubule organizing center through its association with CLASPs. CLASP proteins are required at the trans-Golgi network to anchor the Golgi to the cytoskeleton, as well as attract γ-tubulin, which acts as the seed for growing microtubule networks. CLASPs also maintain established microtubules by binding along their length and stabilizing them to prevent microtubule catastrophe. At the growing plus-end of microtubules, CLASPs bind to attach microtubules to the plasma membrane. If CLASPs ability to perform any of these functions is modified or lost, it results in loss of the Golgi structure and function, catastrophe for the cytoskeleton, and loss of cell polarity. There are multiple supporting proteins that associate with CLASPs and are required for its proper function. The trans-Golgi network specific golgin 40 GCC185 tethers CLASPs to the Golgi anchoring the cytoskeleton. The PAR complex negatively regulates brain specific CLASP2 interactions with GCC185. PAR3 binds directly to CLASP2, bringing the aPKC in range to phosphorylate the C-terminus of CLASP2 localizing it to the plus-end of microtubules. CLASPs have also been shown to be phosphorylated at the C-terminus by GSK3β targeting it to the plus-end of microtubules. This suggests that unphosphorylated CLASPs are localized to the trans-Golgi network. Loss of function to any of a number of CLASP-associating proteins has implications on Golgi organization, cytoskeletal structure, and cell polarity. The brain specific CLASP2 is required for the Golgi apparatus to establish neuronal polarity. Polarity in neurons is essential in establishing proper dendritic arborization and axonal projection. Without these neuron specific structures, neurons are unable to migrate properly during development or establish synapses to relay information. Loss of CLASP2 results in decreased dendritic arborization and axon length; while over expression of CLASP2 results in multiple axons being formed, as well as increased dendritic branching. Both of these phenotypes result in improper neurodevelopment signifying the critical role CLASP2 plays in Golgi regulated neuronal polarity. The Golgi apparatus plays a crucial role in establishing neuronal polarity. Future studies in this area should aim to elucidate the exact mechanisms of how the Golgi apparatus modulates neuronal polarity in primary neurons or in vivo. 41 Much of the work on cell polarity up to this point has been performed in nonneuronal cells. For example, the work on GCC185 interaction with brain specific CLASP2 being modulated by the PAR complex was performed in COS-7 (kidney) cells expressing exogenous CLASP2. It is clear that CLASP2 plays an essential role in establishing neuronal polarity, and that CLASP2 has an intimate relationship with the Golgi apparatus. The next steps should be to perform similar studies with CLASP2 and associated proteins, such as GCC185, in primary cell cultures and in vivo, to further elucidate the mechanism of Golgi-dependent neuronal polarity. 42 JOURNAL ABBREVIATIONS: Abbreviation Annu. Rev. Cell Dev. Biol. Annu. Rev. Neurosci. Biochim. Biophys. Acta. Cell Mol. Life Sci. Cell Rep. Cold Spring Harb. Perspect. Biol. Curr. Opin. Cell Biol. Curr. Opin. Neurobiol. Dev. Cell Front. Cell Neurosci. Front. Neuroanat. Genes Dev. Histochem. Cell Biol. Int. Rev. Cell Mol. Biol. J. Cell Biol. J. Cell Sci. J. Leukoc. Biol. J. Neurosci. Mol. Cell Neurosci. Mol. Biol. Cell Nat. Cell Biol. Nat. Genet. Nat. Rev. Mol. Cell Biol. Neuropsychol. Rev. Plant Physiol. Prog. Biophys. Mol. Biol. Trends Biochem. Trends Cell Biol. Journal Name Annual Review of Cell and Developmental Biology Annual Review of neuroscience Biochimica Et Biophysica Acta Cellular and Molecular Life Sciences Cell Reports Cold Spring Harbor Perspectives in Biology Current Opinion in Cell Biology Current Opinion in Neurobiology Developmental Cell Frontiers in Cellular Neuroscience Frontiers in Neuroanatomy Genes & Development Histochemistry and Cell Biology International Review of Cell and Molecular Biology Journal of Cell and Biology Journal of Cell Science Journal of Leukocyte Biology Journal of Neuroscience Molecular and Cellular Neuroscience Molecular Biology of the Cell Nature Cell Biology Nature Genetics Nature Reviews Molecular Cell Biology Neuropsychology Review Plant Physiology Progress in Biophysics and Molecular Biology Trends in Biochemical Sciences Trends in Cell Biology 43 REFERENCES: Akhmanova, A., and Hoogenraad, C.C. (2005). Microtubule plus-end-tracking proteins: mechanism and functions. Curr. Opin. 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Sci. 118, 2279–2293. Zhu, X., and Kaverina, I. (2013). Golgi as an MTOC: making microtubules for its own good. Histochem. Cell Biol. 140, 361–367. 50 CURRICULUM VITAE: Tyler D. Ash, 810-814-0593, [email protected], 2350 Nadine Drive, Sterling Heights, MI 48825 Education: Boston University, Boston, Massachusetts • Master of Science in Molecular Biology, Cellular Biology, and Biochemistry • Research performed in Molecular Biology of the Neuron Lab Michigan State University, East Lansing, Michigan • Bachelor of Science in Microbiology • Lyman Briggs Residential College for the Study of Science • Minor: History, Philosophy, and Sociology of Science May 2015 May 2013 Research Experience: Graduate Student, Dr. Angela Ho, Cellular and Molecular Neuroscience Laboratory Boston University, Boston, Massachusetts December 2013 – February 2014, July 2014 – May 2015 • Mastered various molecular biology techniques including confocal microscopy, cloning, shRNA knockdown of targeted proteins, infection and transfection of cell cultures, western blot, immunohistochemistry, immunocytochemistry, coimmunoprecipitation , and aseptic subculturing techniques • Worked with mice as models for development • Presented research data as well as journal papers in lab meetings and journal club Laboratory Rotation, Dr. Tarik Haydar, Laboratory of Neural Development Boston University, Boston, Massachusetts • Performed confocal microscopy, cloning, shRNA protein knockdown, western blot, immunocytochemistry, and subculturing techniques • Established N2A cell line from mouse neuroblastoma in the lab May – June 2014 Laboratory Rotation, Dr. Aaron Beeler, Medicinal Chemistry Laboratory March – April 2014 Boston University, Boston, Massachusetts • Performed pull-down assays to identify protein binding partners for drug targets • Exposed to the process of organic synthesis Laboratory Rotation, Dr. Horacio Frydman, Host-Microbe Interactions in Stem Cell Niche Laboratory Boston University, Boston, Massachusetts September – November 2013 • Dissected reproductive organs of Drosophila • Performed confocal microscopy and immunohistochemistry Research Assistant, Dr. Mark Whalon, Alternative Pesticides Laboratory, Michigan State University, East Lansing, Michigan • Wrote portions of federal grant proposals, reports, and policy papers • Performed insect dissections, database management, in vitro bio-assays, grant budget management, and field work June 2010 – August 2013 51 Research Assistant, Dr. Cindy Arvidson, Pathogenesis of Neisseria gonorrheae Laboratory Michigan State University, East Lansing, Michigan September 2011 – May 2013 • Research funded for $1,000 by the National Science Foundation • Performed DNA extraction and transformations • Trained an undergraduate student to perform DNA extractions, gene transformations, and other general lab duties Research Intern, Dr. Douglas Luckie, Genetic Disease Research Laboratory, Michigan State University, East Lansing, Michigan • Pursued, presented, and defended results on a research project aimed at developing a PCR-based diagnostic assay for a mutation known to cause sickle cell anemia • Gained experience with PCR, DNA sequence analysis, primer design, agarose gel electrophoresis with both TBE and rapid Lithium Borate January 2011 – May 2011 Lab Training Completed: Responsible Conduct of Research Training IACUC-Mouse Handling Biosafety Level 2 Office of Radiation, Chemical and Biological Safety February 2015 August 2014 September 2011 June 2010 Honors/Awards: First place award at University Undergraduate Research and Arts Forum (UURAF) Michigan State University • Cell Biology, Genetics, and Genomics for poster presentation of Detection of 1510 BP Band Using PCR and Gel Electrophoresis to Diagnose E6V Sickle Cell Anemia in Human Genomic DNA Lyman Briggs College National Science Foundation S-STEM Scholarship • Awarded $18,000.00 of funding over three years to undergraduates at Michigan State University in Lyman Briggs College with an interest in working in a research related scientific field in the future who have maintained a minimum 3.0 GPA Lyman Briggs College National Science Foundation STEM Research Funding • $1,000.00 of funding was provided in support of research through the Pathogenesis of N. gonorrheae lab to work with gene regulation Activities: Study Abroad: History of Science in Florence and Rome, Italy • Studied the interactions between Galileo and the Roman Catholic Church during the transition from geocentrism to heliocentrism • Examined how scientific discoveries influence societies and cultures Spring 2011 August 2010 July 2012 May – June 2012 52 Skills: • Skilled in grant writing and budget management • Proficient in Zen microscopy software, R statistical package, Adobe InDesign and Photoshop Professional Works: Title Authorship Type of Work Functional Ecology Indicators to Help Defend Michigan Apple Production Practices, 2010 Summary Mark Whalon, Jeanette Wilson, Tyler Ash Poster presented at the Great Lakes Expo, 2010 USDA Risk Avoidance and Mitigation Program in Michigan Tart Cherries: Pest Status, Economics, and Ecological Effects in the Transition from Organophosphates to Reduced Risk IPM Detection of 1510 BP Band Using PCR and Gel Electrophoresis to Diagnose E6V Sickle Cell Anemia in Human Genomic DNA Mark Whalon, Jeanette Wilson, Tyler Ash, Alex Johnson Poster presented at the Entomological Society of America Conference, 2010 Tyler Ash, Jon Walters, Kristina Knirk, Nicole Arcy Poster presented at University Undergraduate Research and Arts Forum (UURAF) at Michigan State University, 2011 Functional Ecology Indicators to Help Defend Michigan Apple Production Practices, 2011 Summary Mark Whalon, Tyler Ash Poster presented at the Great Lakes Expo, 2011 US Tart Cherries: Pesticide Policy and the Precautionary Principle, 22: (4) 177-182. Outlooks on Pest Management Acknowledged Journal Publication, 2011 Risk Avoidance and Mitigation Program in Tart Cherries: a Seven Year Project Multiple authors including Tyler Ash, can be provided upon request United States Department of Agriculture (USDA) Funded Grant Report, 2012 Tart Cherry Pest Management in the Future: Development of a Strategic Plan Multiple authors including Tyler Ash, can be provided upon request Policy Document submitted to the United States Environmental Protection Agency (EPA), 2012