Nuclear Ca2+ regulates many signaling pathways including NFAT, CREB, MEF that are essential to cell immunity, development and differentiation. In current stage, there is no specified biochemical drugs to regulate nuclear Ca2+ individually. The existence of nuclear Ca2+ store remains unknown. In this study, we show the femtosecond-laser stimulation can precisely regulate nuclear and other subcellular Ca2+ stores exclusively. The stimulated Ca2+ store can be released but other subcellular Ca2+ stores are not interfered by femtosecond laser. By using this method, we present the evidence of the endogenous Ca2+ store inside nucleus. The nuclear Ca2+ store volume and release mechanism are clarified. By photostimulation, nuclear Ca2+ store can be released and impact the calcium in endoplasmic reticulum (ER), suggesting a Ca2+ transmission channel between nucleus and ER. The permeability of nuclear pore complex (NPC) to Ca2+ at different Ca2+ levels is also found different. This study indicates the existence of nuclear Ca2+ store and the final mapping of subcellular Ca2+ stores. This result thus is of great significance to the cell calcium theory, Ca2+ signaling, Ca2+-related cell processes, and further other Ca2+-related fields.
Stem-cell therapy is showing great potential in regenerative medicine for their inherent ability to self-renew and differentiate. Biochemical and genetic methods and 3D materials/structures have been developed and making great progress in controlling the development and differentiation of stem cells. One of the major remaining concerns is the invasion of those technologies. In this study, we demonstrate an all-optical and noninvasive approach to precisely induce the differentiation of adipose-derived stem cells (ADSCs) and cerebellar granule neuron progenitor (GNP) cells. We show that the single-time fast-flash of photostimulation by a tightly-focused femtosecond laser, without any optogenetics, can activate endogenous signaling pathways for stem-cell differentiation directly by multiphoton excitation. The significant upregulation of differentiation regulator RUNX2 and Osterix in ADSCs 7 days after photostimulation indicates the osteogenic differentiation of ADSCs. The differentiation was finally confirmed by Alizarin red staining 28 days after photostimulation. The differentiation of GNP cells in vitro and in vivo can also be induced by this method. This noninvasive optical technology hence provides an encouraging advance to activation of signaling pathways in cells and alternative to classic biochemical methods for stem-cell differentiation. This result also provides an optical choice with promising potential for clinical regenerative applications.
Mitochondrial oxidative flashes (mitoflashes) are oxidative burst events in mitochondria. It is crosslinked with numerous mitochondrial molecular processes and related with pivotal cell functions such as apoptosis and respiration. In previous research, mitoflashes were found as spontaneous occasional events. It would be observed more frequently if cells were treated with proapoptotic chemicals. We show that multiple mitoflashes can be initiated by a single femtosecond-laser stimulation that was tightly focused on a diffraction-limited spot in the mitochondrial tubular structure. The mitoflash events triggered by different photostimulations are quantified and analyzed. The width and amplitude of mitoflashes are found very sensitive to stimulation parameters including laser power, exposure duration, and total incident laser energy. This study provides a quantitative investigation on the photostimulated mitoflashes. It may thus demonstrate such optical method to be a promising technique in future mitochondrial research.
Store-operated calcium (SOC) channels, regulated by intracellular Ca2+ store, are the essential pathway of calcium signaling and participate in a wide variety of cellular activities such as gene expression, secretion and immune response1. However, our understanding and regulation of SOC channels are mainly based on pharmacological methods. Considering the unique advantages of optical control, optogenetic control of SOC channels has been developed2. However, the process of genetic engineering to express exogenous light-sensitive protein is complicated, which arouses concerns about ethic difficulties in some research of animal and applications in human. In this report, we demonstrate rapid, robust and reproducible two-photon activation of endogenous SOC channels by femtosecond laser without optogenetics. We present that the short-duration two-photon scanning on subcellular microregion induces slow Ca2+ influx from extracellular medium, which can be eliminated by removing extracellular Ca2+. Block of SOC channels using various pharmacological inhibitors or knockdown of SOC channels by RNA interference reduce the probability of two-photon activated Ca2+ influx. On the contrary, overexpression of SOC channels can increase the probability of Ca2+ influx by two-photon scanning. These results collectively indicate Ca2+ influx through two-photon activated SOC channels. Different from classical pathway of SOC entry activated by Ca2+ store depletion, STIM1, the sensor protein of Ca2+ level in endoplasmic reticulum, does not show any aggregation or migration in this two-photon activated Ca2+ influx, which rules out the possibility of intracellular Ca2+ store depletion. Thereby, we propose this all-optical method of two-photon activation of SOC channels is of great potential to be widely applied in the research of cell calcium signaling and related biological research.
Calcium is an important messenger in cells and whose store and diffusion dynamics at the subcellular level remain unclear. By inducing a controlled slow subcellular Ca2+ release through femtosecond laser irradiation in HeLa cells immersed in different media, cytoplasm is identified to be the major intracellular Ca2+ store, with the nucleus being the minor store and the extracellular Ca2+ also contributing to the total cellular Ca2+ level. Furthermore, Ca2+ released in either the cytoplasm or nucleus diffuses into the nucleus or cytoplasm, respectively, at different rates and influences the Ca2+ release in those regions.
Access to the requested content is limited to institutions that have purchased or subscribe to SPIE eBooks.
You are receiving this notice because your organization may not have SPIE eBooks access.*
*Shibboleth/Open Athens users─please
sign in
to access your institution's subscriptions.
To obtain this item, you may purchase the complete book in print or electronic format on
SPIE.org.
INSTITUTIONAL Select your institution to access the SPIE Digital Library.
PERSONAL Sign in with your SPIE account to access your personal subscriptions or to use specific features such as save to my library, sign up for alerts, save searches, etc.