The authors declare that there are no conflicts of interest present.
Mechanical vibration induces cytoskeletal remodeling, which may influence organelle-associated gene expression. Because Golgi structure depends on cytoskeletal interactions, we tested whether vibration alters mRNA levels of
Vibration is used for medical care, including migraine treatment and neurological disorders (Juto, 2015; Ebersbach, 2008). At the cellular level, vibration has been shown to alter cytoskeletal organization and increase calpain activity, leading to actin remodeling (LaGier et al., 2022). Mechanical cues can regulate gene expression through mechanotransduction pathways (Dupont et al., 2011). Because the Golgi apparatus depends on cytoskeletal interactions for structural integrity, these changes may impact Golgi-associated proteins (Ravichandran et al., 2020; Kulkarni-Gosavi et al., 2019).
To test whether vibration affects expression of a Golgi-associated gene, we measured transcript levels of
HeLa cells were exposed to vibration (~12 mm/s, 15 minutes) or left untreated on a vibration-free table. RNA was extracted, converted to cDNA, and analyzed by qRT-PCR using
We observed reduced
These results indicate that acute (15 minute) vibration reduces expression of a Golgi-associated gene without affecting
Mechanical agitation by vortexing generates vibration by moving fluid parallel to the cells, but it can also introduce heat, which can trigger changes in transcription levels, such as heat shock genes. Therefore, we cannot rule out that localized temperature elevations somehow contributed to altered GOLGA2 levels. To isolate mechanical forces from heat in future studies, temperature monitoring, perhaps with micro-thermocouples, should be performed.
Mechanistically, acute mechanical vibration induces rapid cytoskeletal rearrangement, including calpain-mediated actin cleavage (LaGier et al., 2022). Because Golgi structural integrity relies heavily on intact actin microfilaments and their motors, acute disruption of actin networks may trigger targeted feedback signaling to downregulate key matrix components such as GM130 (
At the cellular level, downregulating
HeLa epithelial cells were seeded into C60 plates on three separate occasions (N=3) and grown to 90% confluency at 37°C in a humidified 5% CO 2 incubator. At this point, HeLa epithelial cells were subjected to mechanical vibration at 12.09 ± 1.69 mm/s vibration for 15 minutes using an analog vortex mixer (V4 condition) at room temperature 20-22°C. Control cells were not vibrated (V0 condition) under identical ambient room temperature condition for 15 minutes.
Total RNA was extracted using the SurePrep™ TrueTotal™ RNA Purification Kit (Fisher Scientific) following manufacturer instructions. RNA concentration was measured using a NanoDrop Lite spectrophotometer. Samples were normalized and reverse transcribed using a High-Capacity RNA-to-cDNA kit.
qRT-PCR was performed using TaqMan® Fast Advanced Master Mix (Applied Biosystems) on a QuantStudio 5 system. Commercial TaqMan® Gene-Expression Assays (ThermoFisher Scientific) were used for
qRT-PCR data was normalized to
HeLa epithelial cells (RRID: CVCL_0030)
SurePrep™ TrueTotal™ RNA Purification Kit (Fisher Scientific)
High-Capacity RNA-to-cDNA Kit
TaqMan® Fast Advanced Master Mix (Applied Biosystems)
TaqMan probes:
QuantStudio 5 Real-Time PCR System
We want to thank the Grand View University Biology Department and The Roy J. Carver Charitable Trust for providing funds to purchase the QuantStudio qPCR equipment and consumables used in this research.
For initial review and critiques, we want to thank the Cell Biology Education Consortium (CBEC): Path to Publication (Award ID #2316122) through the National Science Foundation (NSF).