Apoptosis is an important cellular event that plays a key role in the therapy of many diseases.The mechanism of the initiation and regulation of photodynamic therapy(PDT)–induced apoptosis is complex.Our previous study found that Photofrin was localized primarily in mitochondria,the primary targets of Photofrin-PDT.The key role of Bax in the mitochondria-mediated apoptosis has been demonstrated in many systems.In order to determine the role of Bax in the mitochondrion-mediated apoptosis induced by Photofrin-PDT,we used the GFP-Bax plasmid to monitor the dynamics of Bax activation after PDT treatment.With laser scanning confocal microscopy,we found that Bax did not translocate from the cytosol to mitochondria when the mitochondrial membrane potential(∆Ψm)disappeared,measured by TMRM.Thus,for Photofrin-PDT,the commitment to cell death is independent of Bax activation.
Sirtuins comprise a family of enzymes implicated in the determination of organismal lifespan in yeast and the nematode.Human sirtuin SIRT1 has been shown to deacetylate several proteins in a NADt-dependent manner.It is reported that SIRT1 regulates physiological processes including senescence,fat metabolism,glucose homeostasis,apoptosis,and neurodegeneration.In general,SIRT1 has initially been thought to represent an exclusive nuclear protein.However,depending on the cell lines and organisms examined,a partial or temporary cytoplasmic localization was observed in murine pancreatic beta cells and neonatal rat cardiomyocytes.Since SIRT1 deacetylates both histone and nonhistone-proteins,such as a number of transcription factors,changes in subcellular localization probably play a role in the regulation of its function.In the present studies,we investigated the subcellular localization of SIRT1 in response to growth factor deprivation in African green monkey SV40-transformed kidneyfibroblast cells(COS-7).Using SIRT1-EGFPfluorescence reporter,we found that SIRT1 localized to nucleus in physiological conditions.We devised a model enabling cell senescence via growth factor deprivation and found that SIRT1 partially translocated to cytosol under the treatment,suggesting a reduced level of SIRT1 activity.We found PI3K/Akt pathway was involved in the inhibition of SIRT1's cytosolic translocation,because inhibition of these kinases significantly decreased the amount of SIRT1 maintained in nucleus.Taken together,we demonstrate that growth factor deprivation induces cytosolic translocation of SIRT1,which suggests a possible connection between cytoplasm-localized SIRT1 and the aging process and provides a new application of single moleculefluorescence imaging of the molecule events in living cells.
Microglial activation plays an important role in neurodegenerative diseases.Once activated,they have macrophage-like capabilities,which can be beneficial by phagocytosis and harmful by se-cretion of neurotoxins.However,the resident microglia always fail to trigger an effective pha-gocytic response to clear dead cells or Aβdeposits during the progression of neurodegeneration.Therefore,the regulation of microglial phagocytosis is considered a useful strategy in searchingfor neuroprotective treatments.In this study,our results showed that low-power laser iradiation(LPLI)(20 J/cm²)could enhance microglial phagocytic function in LPS-activated microglia.Wefound that LPLI-mediated microglial phagocytosis is a Rac-1-dependent actin-based process,that a constitutively activated form of Rac1(RaclQ61L)induced a higher level of actin pol-ymerization than cells transfected with wild-type Racl,whereas a dominant negative form ofRacl(RaclT17N)markedly suppressed actin polymerization.In addition,the involvement of Racl activation after LPLI treatment was also observed by using a Raichu fluorescence resonance energy transfer(FRET)-based biosensor.We also found that PI3K/Akt pathway was required inthe LPLI-induced Raci activation.Our research may provide a feasible therapeutic approach tocontrol the progression of neurodegenerative diseases.
