Bio-captured Synthetic Probes For Imaging Of Stressed Organelles

Stressed lysosomes are highly related to a variety of pathological processes.Stressed lysosomes refer to lysosomes whose parameters(pH,size,shape,quantity,location,and membrane permeability,etc.)are changed under external stimulations.For example,lysosomal membrane permeabilization leads to the release of hydrolases into the cytoplasm and then triggers cell death.A variety of lysosomal imaging probes have been developed,but they are often dependent on acidic pH and membrane integrity of lysosomes,making it difficult to monitor stressed lysosomes with increased membrane permeabilization or membrane rupture.Therefore,approaches capable of continual tracking of stressed lysosomes would be useful to decipher the roles of lysosomes in biology and diseases.Lysosomal membrane permeabilization(LMP)engaged in multiple human diseases is accompanied by relocation of cytosolic galectin into LMP+lysosomes.We herein reported a galectin trafficking-targeted method to image LMP using two kinds of glyco-dendrimers,a sialic acid-terminated dendrimer labeled with pH-inert rhodamine and a lactose-terminated dendrimer labeled with fluorescein that becomes green emissive in pH elevated lysosomes.Albeit both accumulated in physiological lysosomes,the former is released from LMP+ lysosomes while the latter binds to galectin accumulated in LMP+lysosomes and thus trapped in LMP+lysosomes.Accordingly,LMP+lysosomes exhibit loss of red fluorescence and turn-on green fluorescence due to loss of lysosomal acidity.This red-to-green color switch enables discernment of LMP+ lysosomes from physiological lysosomes and pH-elevated lysosomes,and can be further utilized to detect LMP in distinct cell death pathways These results suggest the utility of galectin trafficking pathway-integrated synthetic probes for detection of LMP,a key factor for diseased cells.However,lysosomal rupture engaged in diverse diseases is still lack of efficient imaging methods and remains poorly discerned from lysosomal membrane permeabilization(LMP).We herein reported an efficient two-step fluorescence labeling of cysteine cathepsins,which is employed to discern lysosomal rupture by accurately positioning released cathepsins from damaged lysosomes into cytosol.The labeling entails covalent attachment of an azide-tagged suicide substrate(Epo-LeuTyrAz)to the enzyme active site,and subsequent bioorthogonal ligation of the introduced azide with DBCOCM-ROX,a ratiometric pH sensor featuring an acidity-reporting red emissive Xrhodamine-lactam(ROX),blue emissive coumarin(CM),and azide reactive DBCO moiey.Aided with fluorescein isocyanate-labeled sialic acid(FITC-Sia),which is a small molecular lysosomal probe with green fluorescence at neutralized conditions and can be released from LMP+ lysosomes,four states of lysosomes can be discerned by optical imaging:ruptured lysosomes(blue in cytosol),LMP+lysosomes(blue in lysosomes),pH-elevated lysosomes(blue and green in lysosomes)and physiological lysosomes(blue and red in lysosomes).This approach would be useful to study lysosomal rupture over LMP in diseased cells and to evaluate lysosomal ruptureinducing drugs.Distinguishment of newly formed and old lysosomas is of great significance to lysosomal metabolism study.Cathepsin is one of the key proteins in the lysosomal,involved in almost all processes associated with lysosomes.Therefore,we aim to distinguish and monitor the newly formed and old lysosomes by capturing cathepsins in newly formed lysosomes.For this purpose,we synthesized two probes for cysteine cathepsin labeling,Epo-LeuTye-FPEt and Epo-LeuTye-Rox,with different emission wavelengths.These two probes have bright acidity-independent fluorescence and can stably indicate the location of lysosomes with different states.Both probes can covalently label proteases to inactivate them and retain in lysosomes for a long time,which is an important basis for distinguishing lysosome subgroups and performing long-term lysosomal localization imaging.On this basis,for the first time,lysosomes were discerned into two subgroups by cysteine cathepsins labeling,and we preliminarily observed the distribution of newly formed lysosomes in cells through imaging.Furthermore,we observed the process of transfer or exchange of lysosomal cathepsins through fusion or contact between lysosomes.Based on the covalent capture of cathepsin,we basically achieved the labeling and discernment of newly formed lysosomes in living cells.In summary,this research shows that rationally designed small molecule probes can be used for imaging analysis and monitoring of lysosomes under different degrees or states of stress stimulations,which provide valuable tools for the diagnosis and treatment of lysosome related diseases.