Fibroblast PER2 circadian rhythmicity depends on cell density

Area of Science:

• Chronobiology
• Cellular Biology
• Molecular Biology

Background:

• Single fibroblasts, like suprachiasmatic nucleus (SCN) neurons, can act as independent circadian oscillators.
• SCN neurons synchronize via intercellular signaling, but fibroblast synchronization mechanisms remain unclear.
• Previous studies lacked isolated fibroblasts, leaving potential cell-cell interactions unexamined.

Purpose of the Study:

• To investigate the autonomy of single fibroblasts as circadian oscillators.
• To determine if cell density and intercellular signals influence fibroblast circadian rhythmicity.
• To explore the nature of paracrine signals required for fibroblast oscillator function.

Main Methods:

• Single-cell imaging of PER2::LUC reporter mouse fibroblasts in high- and low-density cultures.
• Co-culture experiments mixing PER2::LUC wild-type (WT) cells with nonluminescent Bmal1-/- or Cry2-/- fibroblasts.
• Intervention experiments using conditioned medium, high potassium (K+), or high calcium (Ca2+) to rescue rhythmicity.

Main Results:

• Fibroblast PER2::LUC rhythmicity was significantly reduced in low-density cultures.
• WT fibroblasts maintained rhythmicity in high-density cultures, even when mixed with nonrhythmic Bmal1-/- cells.
• Conditioned medium from high-density cultures rescued rhythmicity in low-density fibroblasts, while high K+ or Ca2+ did not consistently rescue rhythmicity.

Conclusions:

• Fibroblasts require paracrine signals from adjacent cells for robust circadian rhythmicity.
• These essential paracrine signals do not need to be rhythmic themselves.
• Rhythmic signals from other cells do not alter the intrinsic periods of fibroblast oscillators.