Journal: bioRxiv

Article Title: An Atlas of Phosphorylation and Proteolytic Processing Events During Excitotoxic Neuronal Death Reveals New Therapeutic Opportunities

doi: 10.1101/2020.06.15.151456

Figure Lengend Snippet: (A) CRMP2 is cleaved at sites near T509 in its C-terminal tail. Left inset: the abundance (M/L) ratios of the neo-N-terminal peptides at 30 and 240 min after glutamate treatment. Right inset: the abundance ratios of the identified phosphosites in the C-terminal tails of CRMP2 at 30 and 240 min after glutamate treatment. N.D.: not detected. Red scissors: cleavage sites. P in red sphere: phosphorylation. (B) A model depicting the new mechanism of dysregulation of neuronal CRMP2 during excitotoxicity uncovered by our proteomic findings. In control neurons, CRMP2 undergoes hierarchical phosphorylation by Cdk5 and GSK3 at sites in the C-terminal tail. Cdk5 phosphorylates the priming site S522. Upon phosphorylation, pS522 binds GSK3, which catalyses processive phosphorylation of CRMP2 at three other sites in the order of S518, T514 and T509. In excitotoxic neurons, cleavage of CRMP2 generates a long truncated CRMP2 fragment that lacks the priming site S522, abolishing S522 phosphorylation by Cdk5 and in turn suppressing processive phosphorylation of S518, T514 and T509 by GSK3. The truncation and lack of phosphorylation at T509, T514 and S518 may contribute to the accumulation of the immunoreactive CRMP2 signals at the dendritic blebs shown in panel E. (C) Structure of a phosphomimetic mutant of CRMP2 (PDB accession: 5yz5). Dotted line shows the disordered C-terminal tail region. (D) Western blots of lysates from control and glutamate-treated neurons probed with anti-CRMP2, anti-pT509 CRMP2 and tubulin antibodies. Asterisks: potential hyper-phosphorylated forms of intact CRMP2 detected by the anti-CRMP2 and anti-pT509 CRMP2 antibodies. (E) Fluorescence microscopy images showing actin (phalloidin), CRMP2 and nuclei (DAPI) in control and glutamate-treated neurons. White arrows indicate dendritic blebs. The close-up views of the images in the rectangles marked by white dotted lines are shown. Inset: The number of dendritic blebs per mm 2 in control and the glutamate treated neurons in three biological replicates. **: p < 0,01; ***: p <0.001.

Article Snippet: For all experiments, DNA counterstain DAPI (Molecular Probes, Thermo Fisher Scientific) was applied before coverslipping with ProLong gold anti-fade reagent (Invitrogen).

Techniques: Phospho-proteomics, Control, Mutagenesis, Western Blot, Fluorescence, Microscopy

Journal: Water

Article Title: Effects of Underwater Lighting Time on the Growth of Vallisneria spinulosa Yan and Its Water Restoration Process

doi: 10.3390/w16243697

Figure Lengend Snippet: Figure 6. CLSM diagram of leaf–biofilm complex in different light duration ratios. (a) LV: V. spin- ulosa + low light duration ratio, (b) MV: V. spinulosa + medium light duration ratio, and (c) HV: V. spinulosa + high light duration ratio. Red is EPS polysaccharide stained with Texas red, green is protein stained with FITC, and bright blue is DNA stained with DAPI.

Article Snippet: CLSM Determination Plant leaves were collected and cut into small pieces of 5 mm × 5 mm and then placed in a 24−well microplate containing 2.5% glutaraldehyde (0.1 M PBS, pH = 7.2) phosphate buffer for 24 h. After washing 3–5 times with 0.1 M PBS solution (pH = 7.2) (Shanghai Macklin Biochemical Technology Co., Ltd., Shanghai, China), the DNA, extracellular polysaccharides, and proteins were labeled with the fluorescent dyes DAPI, Con A−Texas red conjugate solution (Invitrogen, San Diego, CA, USA), and FITC (Shanghai Macklin Biochemical Technology Co., Ltd., Shanghai, China), respectively.

Techniques: Staining