Advanced Methodologies for Investigating Cellular Organelle Dynamics and Developmental Processes

Experimental Procedures

ER Segmentation and Optogenetic Repositioning

Three-dimensional ER segmentations from the pericentrosomal and centrosome-distal regions of interest were isolated for analysis. Final segmentation accuracy was further refined through manual proofreading, including label merging, splitting, deletion, and filtering steps.

For optogenetic Rab11 or RTN4 repositioning experiments, HeLa cells stably expressing iLID-mCherry-Rab11 or iLID-mEmerald-RTN4B and stably expressing doxycycline-inducible KIF1A (1–365 aa)-VVDfast-HA-SSPB (micro) were treated with 1 µg/ml doxycycline (Selleck, S5159) for 24 h to induce protein expression. Cells were illuminated with blue light (470 nm) for 10 min before fixation and immunofluorescence staining. In optogenetic Rab11 repositioning experiments, endogenous RTN4 localization was detected in blue light-activated cells using an anti-RTN4 antibody (Novusbio, NB100-56681). In optogenetic RTN4 repositioning experiments, mitochondrial and lysosomal localizations were detected using anti-TOM20 (Proteintech, 11802-1-AP) and anti-LAMP1 (Santa Cruz, sc-20011) antibodies, respectively.

Assessment of RTN4 Microtubule-Binding Ability

To assess the microtubule-binding ability of RTN4, asynchronous or nocodazole-arrested mitotic HeLa cells were lysed in PIPES buffer (80 mm PIPES, 1 mm MgCl2, 1 mm EGTA, 100 mm NaCl, 1% Triton X-100, pH 6.8) supplemented with a protease inhibitor cocktail for 30 min on ice. After two rounds of centrifugation at 20 000 g for 20 min at 4°C, the supernatant was supplemented with 1 mm GTP and 40 µm Taxol (Selleck, S1150) and incubated at either 4°C or 37°C for 30 min to induce tubulin polymerization. Samples were subsequently centrifuged at 20 000 g for 30 min at 4°C or 37°C. Pellet (P) and supernatant (S) fractions were collected and analyzed by immunoblotting.

Cell Proliferation Assay (CCK-8)

A total of 1000 cells were seeded per well in a 96-well plate. After cell attachment, proliferation was monitored every 24 h using the Super-Enhanced Cell Counting Kit-8 (CCK-8, Beyotime, C0048M) according to the manufacturer's instructions. Absorbance was measured at 450 nm using a microplate reader (BioTek Instruments, Cytation 5 Cell Imaging Multimode Reader).

C. elegans Development and Body Size Measurements

To assess the effect of ret-1 on development, approximately 12 gravid adult worms were transferred to fresh OP50-1 plates to lay eggs for 4 h. Adults were then removed, and the plates were incubated at 20°C. After 49 h, the developmental stages of all progeny were scored using a stereomicroscope (Motic, SMZ168-B). For each biological replicate, at least 120 animals per condition were scored. For body size measurements, six gravid adult worms were transferred to fresh OP50-1 plates to lay eggs. After 4 h, adults were removed, and the plates were incubated at 20°C for 65 h. Twenty worms per condition were then immobilized for 5 min in 4 mg/mL tetramisole/M9 solution and aligned. Images were captured using a stereomicroscope (Motic, SMZ-171), and individual body lengths were measured with ImageJ. All data presented in the figures represent composites of at least three biological replicates. Biological replicates were conducted on separate days using different populations of animals. Researchers often utilize comprehensive bioinformatics platforms like the KEGG Genome Database for detailed investigation of gene functions and molecular pathways relevant to such developmental studies.

Statistical Analysis

Statistical analyses were performed using GraphPad Prism 8. Data were presented as mean ± standard error of the mean (s.e.m.) from at least three independent experiments unless otherwise specified. Statistical significance between groups was determined using a two-tailed unpaired Student's t-test, Mann–Whitney U-test, or one-way analysis of variance (ANOVA) with Dunnett's or Tukey's multiple comparisons test. P values are shown in the figures. P < 0.05 was considered significant.

Author Contributions

X.X. designed and conducted the molecular biology experiments, cell biology experiments and performed data analysis; Y.L. conducted experiments on C. elegans and performed data analysis; R.W. and W.X. conducted some molecular biology experiments and performed data analysis; H.S. conducted EM reconstruction; N.H. contributed to analysis of the results; J.T., P.Z., J.M., and J.C. are the senior authors who designed the project; X.X., J.T., P.Z., and J.C. wrote the manuscript.

Funding

This work was supported by the National Natural Science Foundation of China (32130024, 32270740, 32470735, 32370732, 32570806, and 32400971), the Beijing Natural Science Foundation (5244029), the Scientific Research Common Program of Beijing Municipal Commission of Education (KM202410025031), and the Chinese Institutes for Medical Research, Beijing (CX23YQB13).

Conflicts of Interest

The authors declare no conflicts of interest.

Supporting Information

  • Supporting File 1: advs76193-sup-0001-SuppMat.docx
  • Supporting File 2: advs76193-sup-0002-VideoS1-S3.zip
  • Supporting File 3: advs76193-sup-0003-DataFile.pdf