Analyzing the role of CtBP1/BARS in neuronal membrane trafficking and polarization

Joint research project

Project leaders: Carmen Valente, Mariano Bisbal
Agreement: ARGENTINA - CONICET - Consejo Nacional de Investigaciones Científicas y Técnicas
Call: CNR/CONICET 2015-2016
Department: Biomedical sciences
Thematic area: Biomedical sciences
Status of the project: New

Research proposal

We aim to study the role of the membrane fission-inducing protein CtBP1/BARS (BARS) in post-Golgi carrier (PGC) formation during neuronal polarization.

BACKGROUND AND SIGNIFICANCE:
Neuronal polarization (NP) is the process of breaking symmetry in new-born nerve cells to create the asymmetry inherent to the formation of the axonal and somatodendritic compartments. This is central to proper brain wiring and for adult brain function. At least three factors underlie NP: extracellular cues, intracellular signaling cascades, and subcellular cytoskeletal/organelle localization-dynamics.
The Argentine Units showed how microtubules (MTs) and actin dynamics at the growth cone serve as a signaling and navigational device for NP. To assure proper neurite extension and differentiation the cytoskeletal organization of the growth cone must be tightly coupled with membrane trafficking. After a sorting step that occurs during budding and fission from the Golgi apparatus (GA) vesicles containing axonal or dendritic proteins are transported by MT- or myosin-based motor transport to their final destinations. Little is known about the elements that control exit from the GA in developing neurons.
The Italian Units have identified the molecular mechanism underlying formation of the carriers that mediate transport from the GA to the plasma membrane (PM). We described a protein complex that comprises BARS and PI4KIII², bridged by a 14-3-3³ dimer, along with ARF, NCS-1 and the PKD and PAK kinases. In this complex BARS binds and activates LPAAT´, which produces PA required for BARS-mediated PGC fission. Notably, 14-3-3³¬, PAK, PKD and LPAAT´ are also involved in BARS-mediated Golgi ribbon unlinking during mitosis, a process also involved in the formation of Golgi outposts in neurons (see aim 2). This study takes advantage of the expertise of the Italian team in the areas of GA organization, exit from the GA and the biology of BARS, and of the Argentinian group on the role of cytoskeletal proteins, MT-based motors and signaling complex in NP.

PRINCIPAL AIMS OF THIS PROPOSAL

1) To evaluate the role of BARS in regulating exit from the GA of neuronal membrane proteins
The Argentinian group will tackle the following questions: Is BARS involved in ER-to-GA or in GA-to-PM of dendritic, axonal or non-polarized neuronal transport? Membrane reporter proteins, already available, will be used. The Italian groups have defined the role of BARS in the basolateral transport in epithelial cells. Thus, as a complementary approach, the RUSH (retention using selective hooks) procedure will be performed in neurons to evaluate if BARS affects selectively ER-to-GA or GA-to-PM transport steps or both. They will take advantage of the already available BARS-related tools in the Italian lab (shRNA, fission-active or inactive mutants, blocking antibody). Preliminary data, in neuronal cells, indicate a role of BARS in exit from the TGN of the transferrin receptor but not of p75NTR. BARS regulates post-Golgi transport in a dynamin-cortactin-actin independent manner. Therefore the Italian group will test if in neurons all types of PGCs generated by BARS are independent on fission pathways that require actin. The role of the BARS-complex components (14-3-3³, PAK, PKD, LPAAT´) in post-Golgi neuronal transport will be analyzed. This neuronal system has never been used by the Italian Unit thus one of its member will visit the Bisbal and Caceres laboratories to learn this technique with the aim to define the BARS machinery and how it is assembled and regulated during neuronal post-Golgi transport. Biochemical approaches and functional neuronal transport assays as well FRET and FLIM-FRET techniques will be used. Preliminary data indicate a role for PKD and PAK in neuronal transport. The BARS-generated PGCs will be analyzed by IF as well as by live cell imaging and correlative light electron microscopy (CLEM). A member of the Argentinean group will visit the Valente lab to learn this technique.

2) To evaluate the role of BARS in the generation of Golgi outposts (GOPs)
In neurons, the GA localizes both the perinuclear region and in dendrites in the form of Golgi outpost (GOPs), which serve as platforms for local delivery of synaptic proteins. After BARS suppression the GOPs disappeared from the dendrites, and tubules originated from the GA. Conversely, BARS expression induced fragmentation of the GA derived tubules and increased the number of dendritic GOPs indicating a role of BARS in GOP generation. The Italian unit will investigate if the BARS-induced Golgi tubulation and GOP dendritic deployment are actin-dependent. The morphology of the GA and GOP formation, will be analyzed in neurons transfected with cofilin- or dynein-GFP (wt or mutants, as actin regulators). In parallel, the Argentinean group will evaluate the role of BARS in the generation of GOPs in minor dendrites. These GOPs seems to be generated locally and not from the central somatic GA. The behavior of GOP formation will be investigated in glicosyltransferases-GFP expressing cells. They will use CLEM for the detailed characterization of GOPs.

3) To evaluate the role of BARS during in situ brain development
Using the in utero electroporation (IUE) method for gene transfer to the embryonic rodent brain the Argentinean group will evaluate the consequence of RNAi suppression of BARS or of expression of BARS wt or fission-active or inactive mutants in cortical development, migration, apical-basal polarity orientation, and distribution of synaptic proteins. Mouse embryos will be IUE at different gestational days and analyzed by confocal microscopy. Preliminary experiments were already performed in Argentine. An Italian member will visit the Bisbal and Caceres labs to learn these techniques.

Research goals

The ability of cells to produce compartmentalized membrane domains and hence to polarize is crucial for complex biological activities, such as the organization of the nervous system. Disruption of the ER-Golgi secretory pathway in developing neurons alters axon-dendritic formation. Therefore, knowledge of the mechanisms underlying exit from the Golgi Apparatus (GA) is crucial for understanding neuronal polarity.

In this proposal we will analyze the role of the protein CtBP1/BARS (BARS) in the formation of post-Golgi carrier and Golgi outposts during neuronal polarization. We aim to:

1) Evaluate the role of BARS in regulating exit from the TGN of axonal and dendritic carriers. We will attempt to characterize whether BARS regulates trafficking of dendritic and/or axonal membrane proteins. If as expected BARS prove to have a role in regulating exit from the GA of neuronal membrane proteins we will explore possible mechanism and its relationship with other elements of the fission machinery as well as the involvement of actin machinery in BARS-mediated exit from the GA.

2) Analyze the participation of BARS in Golgi outposts (GOPs) biogenesis, which serve as platforms for local delivery of post-synaptic receptors during neuronal polarization.

3) Analyze the role of BARS in neuronal development in situ; specifically in cortical neuronal migration and in the polarized orientation of neuritic process within the developing cortex.

Last update: 09/06/2025