DOI: 10.5061/dryad.8sf7m0d0d
title: Data from: Cyclin-dependent kinase 5 (Cdk5) activity is modulated by light and gates rapid phase shifts of the circadian clock

description: The circadian clock enables organisms to synchronize biochemical and
 physiological processes over a 24-hour period. Natural changes in lighting
 conditions, as well as artificial disruptions like jet lag or shift work,
 can advance or delay the clock phase to align physiology with the
 environment. Within the suprachiasmatic nucleus (SCN) of the hypothalamus,
 circadian timekeeping and resetting rely on both membrane depolarization
 and intracellular second-messenger signaling. Voltage-gated calcium
 channels (VGCCs) facilitate calcium influx in both processes, activating
 intracellular signaling pathways that trigger Period (Per) gene
 expression. However, the precise mechanism by which these processes are
 concertedly gated remains unknown. Our study in mice demonstrates that
 cyclin-dependent kinase 5 (Cdk5) activity is modulated by light and
 regulates phase shifts of the circadian clock. We observed that knocking
 down Cdk5 in the SCN of mice affects phase delays but not phase advances.
 This is linked to uncontrolled calcium influx into SCN neurons and an
 unregulated protein kinase A (PKA) – calcium/calmodulin-dependent kinase
 (CaMK) – cAMP response element-binding protein (CREB) signaling pathway.
 Consequently, genes such as Per1 are not induced by light in the SCN of
 Cdk5 knock-down mice. Our experiments identified Cdk5 as a crucial
 light-modulated kinase that influences rapid clock phase adaptation. This
 finding elucidates how light responsiveness and clock phase coordination
 adapt activity onset to seasonal changes, jet lag, and shift work.
description: Mouse behavior, locomotor activity monitoring, light treatment,
 RNA extraction and cDNA synthesis, qRT-PCR, Immunofluorescence, Virus
 production and stereotactic injections, protein extraction,
 Immunoprecipitation, Western blotting, In vitro kinase assay, Cell
 Culture, Fiber photometry, Calcium imaging, FRET imaging
description: # Data from: Cyclin-dependent kinase 5 (Cdk5) activity is modulated by
 light and gates rapid phase shifts of the circadian clock
 [https://doi.org/10.5061/dryad.8sf7m0d0d](https://doi.org/10.5061/dryad.8sf7m0d0d) ## Description of the data and file structure ### Figure 1 1A. The *.PGN files are a graphic representation of the actogram used to analyze and quantify the period length and phase shift measured at different ZTs (14, 24, 10) of scramble and sh Cdk5 mice entrained to light dar cycles 12:12. 1B. The *.pzfx includes the dataset obtained from actograms analyzed with ClockLab. It shows the circadian period length difference between scramble and shCdk5 mice entrained to light-dark cycles 12:12. 1C. The *.pzfx includes the dataset obtained from actograms analyzed with ClockLab. It shows the circadian phase shift difference between scramble and sh Cdk5 mice entrained to light-dark cycles 12:12. 1D. The *.PGN files are a graphic representation of the actogram used to analyze and quantify the period length and phase shift measured at different CTs (14, 24, 10) of scramble and sh Cdk5 mice kept in constant darkness. 1E. The *.pzfx includes the dataset obtained from actograms analyzed with ClockLab. It shows the circadian period length difference between scramble and sh Cdk5 mice kept in constant darkness. 1F. The *.pzfx includes the dataset obtained from actograms analyzed with ClockLab. It shows the circadian phase shift difference between scramble and shCdk5 mice entrained to light kept in constant darkness. ### Figure 2 2A. The folder contains results obtained performing western blots (WB) in SCN samples of wt mice + or - light pulse at ZT14. The folder contains a sub-folder named raw data. The subfolder includes other folders named after the proteins detected by WB. Two *.TIF files are named after the specific protein in each folder. One represents the luminescent signal of the indicated protein, with a protein size marker and the other is without (this one is used for the densitometric quantification). The main folder contains a second *.TIF file, with original blots labeled, where the user can see the protein bands shown in the paper obtained from the original raw data. 2B. The *.pzfx includes the dataset obtained from WB analyzed with ImageJ. It shows the pPKA relative (pPKA/Tubulin) densitometric signal difference in SCN samples of wt mice + or - light pulse at ZT14. 2C The *.pzfx includes the dataset obtained from WB analyzed with ImageJ. It shows the p35 relative (p35/Tubulin) densitometric signal difference in SCN samples of wt mice + or—light pulse at ZT14. 2D. The folder contains results obtained performing western blots (WB) in SCN samples of wt mice + or - light pulse at ZT14. The folder contains a sub-folder named raw data. The subfolder includes: *.TIF coomassie staining, *.ppt_coomassie labeled, *.TIF CDK5 luminescent signal with or without protein size marker, and different autoradiography films. The main folder contains a second *.TIF file, with original blots and autoradiography films labeled, where the user can see the protein bands shown in the paper obtained from the original raw data and for the quantification. 2E. The *.pzfx includes the dataset obtained from WB and autoradiography analyzed with ImageJ. It shows the CDK5 kinase activity (32p-H1/ (IP_CDK5 * 1ug H1 coomassie) densitometric signal difference in SCN samples of wt mice + or - light pulse at ZT14. 2F. The folder contains results obtained performing western blots (WB) in SCN samples of wt mice + or - light pulse at ZT14. The folder contains a sub-folder named raw data. The subfolder includes other folders named after the proteins detected by WB. Two *.TIF files are named after the specific protein in each folder. One represents the luminescent signal of the indicated protein, with a protein size marker and the other is without (this one is used for the densitometric quantification). The main folder contains a second *.TIF file, with original blots labeled, where the user can see the protein bands shown in the paper obtained from the original raw data. ### Figure 3 3A-E. The folder contains results obtained by performing immunofluorescence staining in SCN samples of wt scramble, shCdk5 mice + or - light pulse at ZT14. The folder contains a sub-folder named after the protein immunostained for quantification, containing all the *.TIF images used for reproducibility validation and quantification. Finally, the folder includes the *.pzfx that shows the dataset of quantified immunostained signals in SCN samples of wt mice + or - light pulse at ZT14. ### Figure 4 4B-C. Folder Structure: Each group folder has a trials folder, which includes the raw data file (*.lvm) and the analyzed data files for each recorded mouse (Ch*.mat). File Information: The file list *.xlsx file summarizes the following details: ``` \- Path to the data files \- Group designation \- Trial Information \- Condition (Experimental: Sh Cdk5 or Control: Scramble) \- Recorded channel per mouse (Ch*) ``` Script Dependency: ``` The filelist.xlsx file is also required to run the data analysis scripts. The MATLAB scripts and functions are available at: https://github.com/ZENLabCode. ``` 4D. The folder contains results obtained by performing immunofluorescence staining in SCN samples of wt scramble, shCdk5 mice + or - light pulse at ZT14, which is linked to the immunostained proteins shown in the article. ### Figure 5 5A-B. The *.pzfx includes the dataset obtained from the FRET experiment. 5D-F. The folder contains results obtained by performing immunofluorescence staining in SCN samples of wt scramble, shCdk5 mice + or - light pulse at ZT14. The folder contains a sub-folder named after the protein immunostained for quantification, containing all the *.TIF images used for reproducibility validation and quantification. Finally, the folder includes the *.pzfx dataset that shows the quantified immunostained signal in SCN samples of wt mice + or - light pulse at ZT14. ### Figure 6 6A-J. The *.pzfx (GraphPad Prism) includes the dataset obtained from the rt-qPCR experiment. Each folder is named after the gene analyzed. Supplementary Figures. ### Figure S1 S1A-B. Folders contain results obtained by performing immunofluorescence staining in SCN samples of wt scramble, shCdk5 mice + or - light pulse at ZT14, which is linked to the immunostained proteins shown in the article. S1C. The folder includes *.PGN files showing actograms and *.BMP files showing periodograms obtained by Clock Lab, linked to the actograms showed in S1A-B. S1D. The *.pzfx includes the dataset obtained from actograms analyzed with ClockLab. It shows the circadian period length difference between scramble and shCdk5 mice entrained to light-dark cycles 12:12, before and after the light pulse. S1E. The *.PGN files are a graphic representation of the actogram used to analyze and quantify the period length and phase shift measured at different ZTs (14, 24, 10) of female mice entrained to light dar cycles 12:12. S1F-G. The *.pzfx files include the dataset obtained from actograms analyzed with ClockLab. It shows the circadian period length and phase shift difference between scramble and shCdk5 female mice entrained to light-dark cycles 12:12, before and after the light pulse. ### Figure S2 S2A, C. The folder contains results obtained performing western blots (WB) in SCN samples of wt mice + or - light pulse at ZT14. The folder contains a sub-folder named raw data. The subfolder includes other folders named after the proteins detected by WB. Two *.TIF files are named after the specific protein in each folder. One represents the luminescent signal of the indicated protein, with a protein size marker and the other is without (this one is used for the densitometric quantification). The main folder contains a second *.TIF file, with original blots labeled, where the user can see the protein bands shown in the paper obtained from the original raw data. S2B, D. The *.pzfx files include the dataset obtained from WB analyzed with ImageJ. It shows the pCAMKII (S2B) relative (pCAMKII/CAMKII) and pCREB (S2D) relative (pCREB/CREB) densitometric signal difference in SCN samples of wt mice + or - light pulse at ZT14. S2E. The folder contains *.TIFF file showing a coomassie staining. ### Figure S3 S3A-E. The folder contains results obtained by performing immunofluorescence staining in SCN samples of wt scramble, shCdk5 mice + or - light pulse at ZT14. The folder contains a sub-folder named after the protein immunostained for quantification, containing all the *.TIF images used for reproducibility validation and quantification. Finally, the folder includes the *.pzfx that shows the dataset of quantified immunostained signals in SCN samples of wt mice + or - light pulse at ZT14. ### Figure S4 S4A. The *.PGN files are a graphic representation of the actogram used to analyze and quantify the period length measured at different ZTs (14, 24, 10) of scramble and shCdk5 mice injected with GCAMP7 and entrained to light dar cycles 12:12. S4B. The folder contains *.BMP files showing the periodogram of scramble and shCdk5 mice injected with GCAMP7 and entrained to light dar cycles 12:12. S4C. The *.pzfx files include the dataset obtained from actograms analyzed with ClockLab. It shows the circadian period length difference between scramble and shCdk5 mice injected with GCAMP7 and entrained to light-dark cycles 12:12, before and after the light pulse. S4D-E. Raw data are contained in the folder linked to Figure 4B-C. ### Figure S5 S5A. The *.xlsx includes the dataset obtained from the FRET experiment. S5B-C. The folder contains results obtained performing western blots (WB) in SCN samples of wt mice + or - light pulse at ZT14. The folder contains *.TIF files, with original blots with /without protein size marker. S5D. The folder contains pDARP32 acquisitions obtained by performing immunofluorescence staining in SCN samples of wt scramble, shCdk5 mice + or - light pulse at ZT14, which is linked to the immunostained proteins shown in the article. The folder contains a picture for each channel. S5E. The folder contains a subfolder named pDarp32 for quantification, containing all the *.TIF files used for the quantification, and a *.pzfx file includes the dataset obtained from the quantification. ### Figure S6 The *.pzfx (GraphPad Prism) includes the dataset obtained from the rt-qPCR experiment. Each folder is named after the gene analyzed. Usage notes 1\. *.PGN and *.BMP files, produced and exported using ClockLab, can be opened with Paint. 2\. Graph Pad Prism generates any *.pzfx file. Numbers are placed in appropriate columns, and the user can employ the Prism software and challenge the data with desired statistical tests. 3\. All TIF files showing immunofluorescences are exported by *. Liff files were opened using the Las X software. 4\. Western blot and immunofluorescence quantifications are performed using ImageJ. 5\. *.lvm files are generated by LabVIEW and contain data in a text-based format. The user can use MATLAB to import and work with this data ## Code/software The MATLAB scripts and functions are available at: [https://github.com/ZENLabCode](https://github.com/ZENLabCode)