Question

Look at the figure below, borrowed from Chao et al. (2007), "MeCP2 Controls Excitatory Synaptic Strength by Regulating Glutamatergic Synapse Number." In this experiment, individual neurons were grown in culture such that they only formed synapses on themselves (autapses). Three proteins were visualized (VGLUT1, MAP2, PSD95), under three different conditions: wildtype neurons (WT), MeCP2 knockout neurons (Null), and MeCP2 overexpressing neurons (Tg1). A VOLUT1 MAP2B PSD95 C VGLUT1 MAP2 PSD95 MAP2 DIV 7-9 *** 2.0, T WT ** --- *HH -I-- Null Norm. puncta density T91 28 28 20 20 Null Tg1 Null Tg1 VGLUT1 PSD95 Figure 3. MeCP2 Regulates Glutamater- gic Synapse Numbers (A and B) Representative images of autaptic neurons show colocalization between MAP2 (green) and VGLUT1 (A) (blue) or PSD95 (B) (blue). Scale bar, 10 um. (C) Bar graph shows synaptic density of each marker normalized to WT (dotted line). (D) Representative images showing colocaliza- tion of VGLUT1 (blue) and PSD95 (red), arrow- heads, with MAP2 (green). Scale bar, 5 m. (E-G) Bar graphs show fraction of colocaliza- tion for PSD95(E) and VGLUT1 (F) and synaptic density of VGLUT1-PSD95 colocalized puncta (G). Number of neurons analyzed (n) shown in the bars is the same for WT and corresponding group. Data shown as mean + SEM normalized to WT (dotted line). *p < 0.05, **p<0.01, ***p <0.001. WT Null Tg1 MAP2 VGLUT1 PSD95 DIV 9 DIV 9 DIV 9 *** *HH E- Norm. Fraction of PSD95 M Coloc. to VGLUT1 Norm. Fraction of VGLUT1 TI Coloc. to PSD95 Norm. Synaptic Density VGLUT1-PSD95 1.07 I-- - 14 Null Tg1 Null Tg1 Null Tg1 1. If necessary, do a little background research. First, what are these three proteins they visualized, and second, why were they used in THIS experiment? le; What does visualization of these proteins demonstrate/represent? (3) Protein Why use it? What does it indicate/represent? MAP2 VGLUT1 PSD95 2. In panel D, all three proteins were visualized at the same time using immunocytochemistry. Using primary and secondary antibodies, fill in the table below to propose an experimental set- up for how this was accomplished. Be sure to note where the fluorescent color is attached, and which color is used for each protein. (4.5) Primary antibody Secondary antibody MAP2 VGLUT1

Answer 1

(1)

	Protein	Use and representation
MAP2	Microtubule-associated protein 2	It has a role in microtubule synthesis and assembly.
VGLUT1	Vesicular glutamate transporter 1	Upload glutamate into synaptic vesicles
PSD95	Postsynaptic density protein 95	It plays an important role in learning and memory formation