The primary cilium is an organelle that serves as a signaling center of the cell and is involved in the hedgehog signaling, cAMP pathway, Wnt pathways, etc. Ciliary function relies on the transportation of molecules between the primary cilium and the cell, which is facilitated by intraflagellar transport (IFT). IFT88, one of the important IFT proteins in complex B, is known to play a role in the formation and maintenance of cilia in various types of organisms. The ciliary transition zone (TZ), which is part of the gating apparatus at the ciliary base, is home to a large number of ciliopathy molecules. Recent studies have identified important regulating elements for TZ gating in cilia. However, the architecture of the TZ region and its arrangement relative to intraflagellar transport (IFT) proteins remain largely unknown, hindering the mechanistic understanding of the regulation processes. One of the major challenges comes from the tiny volume at the ciliary base packed with numerous proteins, with the diameter of the TZ close to the diffraction limit of conventional microscopes. Using a series of stimulated emission depletion (STED) superresolution images mapped to electron microscopy images, we analyzed the structural organization of the ciliary base. Subdiffraction imaging of TZ components defines novel geometric distributions of RPGRIP1L, MKS1, CEP290, TCTN2 and TMEM67, shedding light on their roles in TZ structure, assembly, and function. We found TCTN2 at the outmost periphery of the TZ close to the ciliary membrane, with a 227±18 nm diameter. TMEM67 was adjacent to TCTN2, with a 205±20 nm diameter. RPGRIP1L was localized toward the axoneme at the same axial level as TCTN2 and TMEM67, with a 165±8 nm diameter. MKS1 was situated between TMEM67 and RPGRIP1L, with an 186±21 nm diameter. Surprisingly, CEP290 was localized at the proximal side of the TZ close to the distal end of the centrin-labeled basal body. The lateral width was unexpectedly close to the width of the basal body, distant from the potential Y-links region of the TZ. Moreover, IFT88 was intriguingly distributed in two distinct patterns, forming three puncta or a Y shape at the ciliary base found in human retinal pigment epithelial cells (RPE), human fibroblasts (HFF), mouse inner medullary collecting duct (IMCD) cells and mouse embryonic fibroblasts (MEFs). We hypothesize that the two distribution states of IFT88 correspond to the open and closed gating states of the TZ, where IFT particles aggregate to form three puncta when the gate is closed, and move to form the branches of the Y-shape pattern when the gate is open. Two reservoirs of IFT particles, correlating with phases of ciliary growth, were localized relative to the internal structure of the TZ. These subdiffraction images reveal unprecedented architectural details of the TZ, providing a basic structural framework for future functional studies. To visualize the dynamic movement of IFT particles within primary cilia, we further conducted superresolution live-cell imaging of IFT88 fused to EYFP in IMCD cells. Our findings, in particular, show IFT88 particles pass through the TZ at a reduced speed by approximately 50%, implying the gating mechanism is involved at this region to slow down IFT trafficking. Finally, we report the distinct transport pathways of IFT88 and Smo (Smoothened), an essential player to hedgehog signaling, to support our hypothesis that two proteins are transported in different mechanisms at the ciliary base, based on dual-color superresolution imaging.

The neonatal subventricular zone (SVZ) is a site of continued postnatal neurogenesis, and is the source of cortical glial cells. Apoptosis is an endogenous process of cell destruction, and is a key event in the proper development of the SVZ. Despite its importance, there is still a lack of knowledge regarding the temporal and spatial occurrence of neonatal SVZ apoptosis, cell types affected and the underlying intrinsic and extrinsic mechanisms that guide the process. This thesis addresses these issues, and in addition, finds a nontraditional mode of neurotrophic action for cell survival in the neonatal SVZ. We assessed SVZ apoptosis by subregion, employing the cell death markers, pH2ax and cleaved caspase 3. The medial SVZ contained the highest density of dying cells at p0, while at p7 there was no significant difference in the apoptotic cell density distribution in the SVZ subregions. Combining cell type specific markers with the death markers used, revealed immature postmitotic neurons were the primary cell type cleared in the p0 medial SVZ. The majority of dying cells in the p7 dorsolateral SVZ (SVZdl) were unable to be identified. Using stereotactic injection of a GFP expressing lentivirus, we determined the p0 medial SVZ cell population to be migratory cells bound for the olfactory bulb. An investigation into the intrinsic and extrinsic mechanisms mediating cell death in the neonatal SVZ, showed BH3-only protein Bim expression in the p0 and p7 SVZ, as well as significantly decreased p0 medial SVZ apoptosis in Bim knockout mice. Bim knockout mice did not show a significant change in apoptosis in the p7 SVZdl. TrkB knockout mice have shown a survival role for the receptor in the lateral ganglionic eminence of the neonatal SVZ. To test this in the p0 medial SVZ using a more specific method, a TrkB blocking antibody was injected into the p0 medial SVZ. This resulted in a significantly higher number of apoptotic cells in the p1 medial SVZ versus controls. These studies demonstrate the dynamic nature of the SVZ with its changing density and identity of apoptotic cells within the subregions. It has also shown the influence of Bim and TrkB signaling in neonatal SVZ apoptosis and survival. Finally, it has identified a premigratory cell population in the p0 medial SVZ, whose survival is mediated by neurotrophin signaling at their site of origin.

The kinase mechanistic target of rapamycin (mTOR) is a regulator of cell growth and survival, protein synthesis-dependent synaptic plasticity, and macroautophagic degradation of cellular components. When active, mTOR induces protein translation and inhibits the protein and organelle degradation process of macroautophagy. Accordingly, when blocking mTOR activity with rapamycin, protein translation is blocked and macroautophagy is induced. In the literature, the effects of rapamycin are usually attributed solely to modulation of protein translation, and not macroautophagy. Nevertheless, mTOR also regulates synaptic plasticity directly through macroautophagy, and neurodegeneration may occur when this process is deficient. Macroautophagy degrades long-lived proteins and organelles via sequestration into autophagic vacuoles, and has been implicated in several human diseases including Alzheimer's, Huntington's and Parkinson's disease. Mice conditionally lacking autophagy-related gene (Atg) 7 function have been exploited to investigate the role of macroautophagy in particular mouse cell populations or entire organs. These studies have revealed that the ability to undergo macroautophagic turnover is required for maintenance of proper neuronal morphology and function. It remained unknown, however, whether it also modulates neurotransmission. We used the Atg7-deficiency model to explore the role of macroautophagy in two sites of the basal ganglia; 1) the dopaminergic neuron, and 2) the direct pathway medium spiny neuron. Briefly, we treated mice with rapamycin, and then examined whether an observed effect was present in control animals, but absent in macroautophagy-deficient lines. We found that rapamycin induces formation of autophagic vacuoles in striatal dopaminergic terminals, and that this is associated with decreased tyrosine hydroxylase (TH)+ axonal profile volumes, synaptic vesicle numbers, and evoked dopamine (DA) release. On the other hand, evoked DA secretion was enhanced and recovery was accelerated in transgenic animals in which the ability to undergo macroautophagy was eliminated in dopaminergic neurons by crossing a mouse line expressing Cre recombinase under the control of the dopamine transporter (DAT) promoter with another in which the Atg7 gene was flanked by loxP sites. Rapamycin failed to decrease evoked DA release or the number of dopaminergic synaptic vesicles per terminal area in the striatum of these mice. Our data demonstrated that mTOR inhibition, specifically through induction of macroautophagy, can rapidly alter presynaptic structure and neurotransmission. We then focused on elucidating the role of macroautophagy in dopaminoceptive neurons, the DA 1 receptor (D1R)-expressing medium spiny neuron. Mice were confirmed to be D1R-specific conditional macroautophagy knockouts as assessed by p62 aggregate accumulation in D1R-rich brain regions (striatum, prefrontal cortex, and the anterior olfactory nuclei), and by analysis of colocalization of Cre recombinase and substance P. Marked age-dependent differences in the presence of p62+ aggregates were noted when comparing the dorsal vs. ventral striatum, and at different ages. We found that the size of striatal postsynaptic densities (PSDs) are modulated by Atg7, as mutant mice have significantly larger PSDs. Surprisingly, we also observed an increase in DAT immunolabel in the dorsal striatum, which suggests that apart from increasing synaptic strength, lack of macroautophagy in postsynaptic neurons could indirectly lead to functional consequences in presynaptic dopaminergic function. Given the newly elucidated role of macroautophagy in modulating a number of pre- and post- synaptic properties, we then explored the potential implications of this process in mediating the effects of synaptic plasticity, specifically to that induced by recreational drugs. An array of studies demonstrates that drugs of abuse induce numerous forms of neuroplasticity in the basal ganglia. Among these changes, rodents that are chronically treated with psychostimulants show increases in dendritic spine density in striatal medium spiny neurons. Little is known about the molecular mechanisms underlying medium spiny neurons gaining more spines in response to psychostimulants. Also, most data, such as involvement of both the D1R and N-methyl-D-aspartic acid (NMDA) receptors, stems from studies using cocaine, and not amphetamine, although a single injection of cocaine has been shown to increase medium spiny neuron spine density, whether acute amphetamine is capable to do so remains to be elucidated. This is an attractive avenue of research to follow given that amphetamines are used recreationally, abused, but unlike cocaine, prescribed for attention deficit hyperactivity disorder and narcolepsy (reviewed in Heal et al., 2013). A myriad of studies has implicated these two proteins in spinogenesis, spine maturation and maintenance, and neuroplasticity. In addition, several studies have demonstrated an association between levels of PSD95 and spine density in various brain regions. Before characterizing the role of mTOR and macroautophagy in psychostimulant-induced plasticity, we examined if an acute injection of amphetamine at multiple doses (1-30 mg/kg) and times of collection after treatment (1-48 hr) influences PSD95 and Homer1b/c in the striatum of wild-type mice by western blotting. We found that amphetamine failed to robustly modify levels of either protein in the striatum. Our data raises several possibilities, including the possibility that unlike cocaine, acute regimens of amphetamine might not regulate spine density in the striatum, and that, it is crucial to examine their effects separately. Finally, this work now provides a starting point to undertake the study of how acute amphetamine affects macroautophagic machinery that regulates molecular, morphological, functional and whole animal behavior.

This work discusses both mechanisms underlying mitochondrial quality control and cytokinesis in the budding yeast Saccharomyces cerevisiae. As these topics are quite different, their presentation has been divided into two parts, "Part I: Mitochondrial Remodeling Through the Proteasome is Critical for Mitochondrial Quality Control in Budding Yeast" and "Part II: Aim44p Regulates Phosphorylation of Hof1p to Promote Contractile Ring Closure During Cytokinesis in Budding Yeast." In Part I, we show that the proteasome is critical for cellular fitness in response to chronic, low levels of mitochondrial reactive oxygen species (ROS) in budding yeast. Deleting DOA1, which is required for ubiquitin-mediated degradation, UFD5, which promotes proteasome gene expression, or NAS2, which promotes proteasome regulatory particle assembly, increases the sensitivity of yeast to chronic, low levels of mitochondrial ROS. In contrast, deleting ATG32, a gene required for mitophagy, other autophagy genes, non-essential chaperones including prohibitins, or mitochondrial proteins including the Lon protease (Pim1p) or YME1, does not affect cellular fitness under these conditions. Doa1p binds with Cdc48p and Vms1p, which associates with mitochondria and promotes extraction of ubiquitinated proteins from the organelle for proteasomal degradation in a pathway called mitochondria-associated degradation (MAD). Elevated mitochondrial ROS increases protein ubiquitination, ubiquitination of the mitochondrial protein aconitase and expression of key MAD proteins. Interestingly, down-regulating ER-associated degradation (ERAD), which shares some common proteins with MAD, can promote cell growth under conditions of elevated mitochondrial ROS. Finally, deletion of DOA1 results in increased sensitivity of yeast and yeast mitochondria to oxidative stress. Mitochondria in doa1 null cells are more oxidized than mitochondria in wild-type or atg32 null cells under conditions of elevated mitochondrial ROS. Moreover, deletion of DOA1 results in a decrease in chronological lifespan. These findings support a critical role for the proteasome and MAD in mitochondrial quality control, which in turn affects cellular fitness, in response to chronic, low levels of mitochondrial ROS. In Part II, we show that the protein product of YPL158C, Aim44p, undergoes septin-dependent recruitment to the site of cell division. Aim44p co-localizes with Myo1p, the type II myosin of the contractile ring, throughout most of the cell cycle. The Aim44p ring does not contract when the actomyosin ring closes. Instead, it forms a double ring that associates with septin rings on mother and daughter cells after cell separation. Deletion of AIM44 results in defects in contractile ring closure. Aim44p co-immunoprecipitates with Hof1p, a conserved F-BAR protein that binds both septins and type II myosins and promotes contractile ring closure. Deletion of AIM44 results in a delay in Hof1p phosphorylation, and altered Hof1p localization. Finally, overexpression of Dbf2p, a kinase that phosphorylates Hof1p and is required for re-localization of Hof1p from septin rings to the contractile ring and for Hof1p-triggered contractile ring closure, rescues the cytokinesis defect observed in aim44 null cells. Our studies reveal a novel role for Aim44p in regulating contractile ring closure through effects on Hof1p.

Vesicular membrane fusion involves the release of contents in a broad array of biological systems, such as intracellular trafficking, secretion, fertilization, and development. It is also a critical step in the infection of cells by membrane enveloped viruses such as HIV, influenza, and Ebola. SNARE proteins form the core of the fusion machinery in nearly all intracellular fusion processes. The initial complete connection between two fusing membranes is the fusion pore. There is considerable evidence that both the fusion machinery and the biophysical properties of the membranes themselves affect contents release, lipid mixing, and fusion kinetics, but the mechanisms are poorly understood. Flickering of fusion pores during exocytotic release of hormones and neurotransmitters is well documented, but without assays that use biochemically defined components and measure single pore dynamics the contributions from different influences are almost impossible to separate. This thesis examines the biophysical mechanisms by which SNAREs and lipid composition control fusion rates and fusion pore kinetics. First, we studied fusion pore flickering in vitro. We used total internal reflection fluorescence (TIRF) microscopy to quantify fusion pore dynamics in vitro and to separate the roles of SNARE proteins and lipid bilayer properties. To interpret the experimental measurements quantitatively, we developed a mathematical model to describe the diffusion of labelled lipids from a vesicle, through a flickering fusion pore, and into a supported bilayer. When small unilamellar vesicles (SUV) bearing neuronal v SNAREs fused with planar bilayers (SBL) reconstituted with cognate t SNARES, lipid transfer rates were severely reduced, suggesting that pores flickered. We developed an algorithm which included a complete description of fluorophores in the TIRF field. We accounted for the intensity decay of the evanescent TIRF wave normal to the SBL, the polarization of the evanescent TIRF wave, and any potential quenching effects. In general, the first two effects are coupled. This algorithm allowed us to measure the sizes of docked vesicles using fluorescent microscopy. From the lipid release times we used the model to compute pore openness, the fraction of the time the pore is open, which increased dramatically with cholesterol. For most lipid compositions tested SNARE mediated and non specifically nucleated pores had similar openness, suggesting that pore flickering was controlled by lipid bilayer properties. However, with physiological cholesterol levels SNAREs substantially increased the fraction of fully open pores and fusion was so accelerated that there was insufficient time to recruit t SNAREs to the fusion site, consistent with t SNAREs being pre clustered by cholesterol into functional docking and fusion platforms. Our results suggest that cholesterol opens pores directly by reducing the fusion pore bending energy, and indirectly by concentrating a number of SNAREs into individual fusion events. In the second part of the thesis, I describe my contributions to a project in which a mathematical model was developed to describe the behavior of SNAREpins connecting SUVs of different sizes to a planar membrane. It was necessary to quantify the membrane membrane and SNAREpin membrane interaction forces. By combining the well known van der Waals, electrostatic, and steric hydration membrane forces with the SNAREpin membrane electrostatic interactions I developed a complete description of the membrane forces involved in SUV-SBL fusion. We then combined the description of the interactions with experimentally measured SNARE zippering energies. We find that the predominant driving forces for membrane fusion, once the SNAREpins have completely zippered, are steric hydration forces among the SNAREpins and membranes. These forces enlarge a SNAREpin cluster, which in turns pulls the membranes together due to curvature effects.

Mechanosensation, the ability for cells to sense and respond to physical cues, is a ubiquitous process among living organisms and its dysfunction can lead to devastating diseases, including atherosclerosis, osteoporosis, and cancer. The primary cilium is a solitary, immotile organelle that projects from the surface of virtually every cell in the human body and can function as a mechanosensor across diverse biological contexts, deflecting in response to fluid flow, pressure, touch and vibration. It can detect urinary flow rate in the kidney, monitor bile flow in the liver, and distinguish the direction of nodal flow in embryos. In this thesis, we examined the interplay of biology and mechanics in the context of this multifunctional sensory organelle from the tissue to subcellular scale. In the first part of this work, we examined the cilium at the tissue level. Primary cilia are just beginning to be appreciated in bone with studies recently reporting loss of cilia results in defects in skeletal development and adaptation. We disrupted primary cilia in osteocytes, the principal mechanosensing cells in bone, and demonstrated that loss of primary cilia in osteocytes impairs load-induced bone formation. Over the course of our work with primary cilia, we also identified the need for more standardized imaging approaches to the cilium and presented an improvement to distinguishing proteins within the cilium from the rest of the cell. In the later part of this work, we examined the primary cilium at the subcellular level. While deflection is integral to the cilium's mechanosensory function, it remains poorly understood and characterized. Using a novel experimental and computational approach to capture and determine the mechanical properties of the cilium, we demonstrated cilium deflection can be mechanically and chemically modulated. We revealed a mechanism, acetylation, through which this mechanosensor can adapt and regulate overall cellular mechanosensing. By modifying our combined experimental and computational approach, we analyzed cilium deflection in vivo for the first time. Collectively, this work uncovers new insights across biological scales in the primary cilium as an extracellular nexus integrating mechanical stimuli and cellular signaling. Understanding the mechanisms driving cilium mechanosensing has broad reaching implications and unlocks the cilium's potential as a therapeutic target to treat impaired cellular mechanosensing critical to a multitude of diseases.

Organelles sequester biological phenomena within the cell, and allow an additional layer of complexity to life. The presence and maintenance of these organelles is crucial for cellular function. Two of the most expansive and complex organelles are the mitochondria and endoplasmic reticulum. These organelles contribute energy, protein folding and secretion, lipids, calcium regulation, and various other metabolites to the biology of the cell. Importantly, these organelles accumulate damage and cannot be derived de novo, therefore must be inherited and maintained in a functioning state. The study of these organelle quality control processes serves as the basis for my thesis. We use the budding yeast as a model organism to uncover conserved pathways affecting organelle, and ultimately cellular homeostasis. In yeast we find mitochondrial inheritance is critical for cell survival. Furthermore, not only is inheritance critical, but inheritance of a certain threshold of functional mitochondria appears critical in maintaining normal lifespan in yeast, identifying mitochondria as an aging determinant. By examining mutants that negatively affect mitochondrial inheritance in yeast, we established a role for phosphatidylcholine biosynthesis in organelle maintenance and inheritance. Glycerophospholipid biosynthesis plays a clear role not only in mitochondrial inheritance but also in that of the endoplasmic reticulum. We use insights gained from yeast to guide research into a human disease caused by similar glycerophospholipid biosynthetic deficiency.

In eukaryotic cells, proteins continuously flux through the organelles of the secretory pathway in an essential cellular process called protein secretion. This dynamic process originates at the endoplasmic reticulum (ER), where translating ribosomes push linear peptides into the ER membrane and lumen. ER chaperones assist in folding nascent peptides into three-dimensional conformations and proteins are concentrated into membrane-encapsulated vesicles bound for the Golgi apparatus. ER to Golgi transport is mediated by a set of cytosolic coat proteins called COPII. The COPII coat polymerizes into a lattice on the ER membrane that is able to bend the membrane around secretory cargo and bud off a spherical vesicle. Protein secretion is subject to rapid changes as a cell responds to its environment and requirements for viability alter. In addition to accommodating short-term demands, such as translational up-regulation, evolved complexity of secretory proteins over time, has also required that secretory components adapt. In both cases changes in secretory demands require that the COPII proteins have an inherent flexibility to navigate these changes without disrupting secretory flux. In this work I have examined a family of quintessential secretory cargo, p24 proteins, that challenge protein secretion. This family of proteins forms a hetero-tetrameric complex that cycles between the ER and the Golgi and mediates transport of glycosylphosphatidylinositol-anchored proteins (GPI-APs). Here I present evidence that suggests, when present in vesicles, both p24 proteins and their GPI-AP cargo present a challenge to vesicle formation. I posit that three attributes of these proteins present a local barrier to membrane bending: Lumenal asymmetric distribution across the membrane, high cellular abundance and affinity for ceramide rich membranes. I have also elucidated mechanisms that the coat has evolved to accommodate troublesome cargo such as p24 proteins, which enhance structural scaffolding and increase average vesicle size. Finally I present preliminary findings indicating that p24s also contribute to ER homeostasis by preventing aberrant incorporation of proteins into vesicles. Comprehensively, these findings have shed light on the role of p24 proteins in vesicles. Traditionally thought to be canonical ER cargo receptors, these proteins also appear capable of contributing to the composition of the vesicles in which they reside, and impacting trafficking efficiency in two ways: First by directly mediating transport of GPI-APs and second by uniformly packing vesicles to avoid wasteful secretion. My work has contributed to a growing notion in the field that secretory cargo are not inert passengers but active participants in vesicle mediated secretion.

Specific regions within the adult mammalian brain maintain the ability to generate neurons. The largest of these, the ventricular-subventricular zone (V-SVZ), comprises the entire lateral wall of the lateral ventricles. Here, a subset of glial fibrillary acid protein (GFAP)-positive astrocytes (B cells) gives rise to neurons and oligodendrocytes throughout life. This process of neurogenesis involves quiescent B cells becoming proliferative (epidermal growth factor receptor (EGFR)-positive) and giving rise to neuroblasts via transit amplifying precursors. The neuroblasts then migrate through the rostral migratory stream (RMS) to the olfactory bulbs (OBs), where they mature into neurons. Studying the stem cells in the V-SVZ has been hindered by the shortage of molecular markers to selectively target them. Using microarray and qPCR analysis of putative quiescent neural stem cells we determined that they were enriched for PDGFRβ mRNA. We used immunostaining to determine the in vivo identity of PDGFRβ+ cells, and discovered that only GFAP+ cells within the V-SVZ stem cell lineage express PDGFRβ. Moreover, these PDGFRβ+ B cells contact the ventricle at the center of ependymal pinwheel structures and the vast majority of them are EGFR-. Importantly, the V-SVZ/RMS/OBcore axis was highly enriched for PDGFRβ expression compared with other brain regions. Detailed morphological analyses of PDGFRβ+ B cells revealed primary cilia at their apical process in contact with the ventricle and long radial processes contacting blood vessels deep within the V-SVZ, both of which are characteristics of adult neural stem cells. When PDGFRβ+ cells were lineage traced in vivo they formed olfactory bulb neurons. Using fluorescence-activated cell sorting (FACS) to purify PDGFRβ+ astrocytes we discovered this receptor is expressed by all adult V-SVZ neural stem cells, including a novel population of EGFR+ PDGFRβ+ cells which correspond to the activated neural stem cells. RNA-sequencing analysis of the purified populations revealed that PDGFRβ+ EGFR+ cells possess a transcriptional profile intermediate between quiescent neural stem cells and actively proliferating GFAP- progenitor cells. Finally, when PDGFRβ is deleted in adult GFAP+ NSCs we observe a decrease in EGFR+ and Dcx+ progenitor cells, together with an increase in quiescent GFAP+ astrocytes. A larger proportion of these mutant cells come in contact with the ventricular lumen, suggesting that PDGFRβ is required for V-SVZ astrocytes to act as stem cells, possibly by mediating interactions with their niche. Taken together, these data identify PDGFRβ as a novel marker for adult V-SVZ neural stem cells that is an important regulator of their stem cell capabilities.

Despite the identification of MYCN amplification as an adverse prognostic marker in neuroblastoma, no drugs that target MYCN have yet been developed. Here, by combining a whole genome shRNA library screen and Master Regulator Inference Algorithm (MARINa) analysis, we identified Transcription Factor Activating Protein 4 (TFAP4) as a novel synthetic lethal interactor with MYCN amplification in neuroblastoma. Silencing TFAP4 selectively inhibits MYCN amplified neuroblastoma growth both in vitro and in xenograft mice models. TFAP4 expression is inversely correlated with patient survival in MYCN-high neuroblastoma. Mechanistically, silencing TFAP4 induces neuroblastoma differentiation, as seen by increased neurite outgrowth, and up-regulation of neuronal markers. TFAP4 regulates a downstream signature similar to the signature of the oncogene anaplastic lymphoma kinase (ALK). Taken together, our results validate TFAP4 as an important master regulator in MYCN amplified neuroblastoma and a novel synthetic interactor with MYCN amplification. Thus, TFAP4 may be a novel drug target for neuroblastoma treatment.