SMN regulates axonal local translation via miR-183/mTOR pathway.
Kye, M.J., Niederst, E.D., Wertz, M.H., Goncalves, I.D., Akten, B., Dover, K.Z., Peters, M., Riessland, M., Neveu, P., Wirth, B., Kosik, K.S., Sardi, S.P., Monani, U.R., Passini, M.A. & Sahin, M.
Hum Mol Genet. 2014 Jul 4. pii: ddu350.
Reduced expression of SMN protein causes spinal muscular atrophy (SMA), a neurodegenerative disorder leading to motor neuron dysfunction and loss. However, the molecular mechanisms by which SMN regulates neuronal dysfunction are not fully understood. Here, we report that reduced SMN protein level alters miRNA expression and distribution in neurons. In particular, miR-183 levels are increased in neurites of SMN-deficient neurons. We demonstrate that miR-183 regulates translation of mTor via direct binding to its 3' UTR. Interestingly, local axonal translation of mTor is reduced in SMN-deficient neurons, and this can be recovered by miR-183 inhibition. Finally, inhibition of miR-183 expression in the spinal cord of an SMA mouse model prolongs survival and improves motor function of Smn-mutant mice. Together, these observations suggest that axonal miRNAs and the mTOR pathway are previously unidentified molecular mechanisms contributing to SMA pathology.
Transcriptome profiling of the demosponge Amphimedon queenslandica reveals genome-wide events that accompany major life cycle transitions.
Conaco, C., Neveu, P., Zhou, H., Arcila, M.L., Degnan, S.M., Degnan, B.M. & Kosik, K.S.
BMC Genomics. 2012 May 30;13:209.
BACKGROUND: The biphasic life cycle with pelagic larva and benthic adult stages is widely observed in the animal kingdom, including the Porifera (sponges), which are the earliest branching metazoans. The demosponge, Amphimedon queenslandica, undergoes metamorphosis from a free-swimming larva into a sessile adult that bears no morphological resemblance to other animals. While the genome of A. queenslandica contains an extensive repertoire of genes very similar to that of complex bilaterians, it is as yet unclear how this is drawn upon to coordinate changing morphological features and ecological demands throughout the sponge life cycle. RESULTS: To identify genome-wide events that accompany the pelagobenthic transition in A. queenslandica, we compared global gene expression profiles at four key developmental stages by sequencing the poly(A) transcriptome using SOLiD technology. Large-scale changes in transcription were observed as sponge larvae settled on the benthos and began metamorphosis. Although previous systematics suggest that the only clear homology between Porifera and other animals is in the embryonic and larval stages, we observed extensive use of genes involved in metazoan-associated cellular processes throughout the sponge life cycle. Sponge-specific transcripts are not over-represented in the morphologically distinct adult; rather, many genes that encode typical metazoan features, such as cell adhesion and immunity, are upregulated. Our analysis further revealed gene families with candidate roles in competence, settlement, and metamorphosis in the sponge, including transcription factors, G-protein coupled receptors and other signaling molecules. CONCLUSIONS: This first genome-wide study of the developmental transcriptome in an early branching metazoan highlights major transcriptional events that accompany the pelagobenthic transition and point to a network of regulatory mechanisms that coordinate changes in morphology with shifting environmental demands. Metazoan developmental and structural gene orthologs are well-integrated into the expression profiles at every stage of sponge development, including the adult. The utilization of genes involved in metazoan-associated processes throughout sponge development emphasizes the potential of the genome of the last common ancestor of animals to generate phenotypic complexity.
Pro-neural miR-128 is a glioma tumor suppressor that targets mitogenic kinases.
Papagiannakopoulos, T., Friedmann-Morvinski, D., Neveu, P., Dugas, J.C., Gill, R.M., Huillard, E., Liu, C., Zong, H., Rowitch, D.H., Barres, B.A., Verma, I.M. & Kosik, K.S.
Oncogene. 2012 Apr 12;31(15):1884-95. doi: 10.1038/onc.2011.380. Epub 2011 Aug29.
MicroRNAs (miRNAs) carry out post-transcriptional control of a multitude of cellular processes. Aberrant expression of miRNA can lead to diseases, including cancer. Gliomas are aggressive brain tumors that are thought to arise from transformed glioma-initiating neural stem cells (giNSCs). With the use of giNSCs and human glioblastoma cells, we investigated the function of miRNAs in gliomas. We identified pro-neuronal miR-128 as a candidate glioma tumor suppressor miRNA. Decreased expression of miR-128 correlates with aggressive human glioma subtypes. With a combination of molecular, cellular and in vivo approaches, we characterize miR-128's tumor suppressive role. miR-128 represses giNSC growth by enhancing neuronal differentiation. miR-128 represses growth and mediates differentiation by targeting oncogenic receptor tyrosine kinases (RTKs) epithelial growth factor receptor and platelet-derived growth factor receptor-alpha. Using an autochthonous glioma mouse model, we demonstrated that miR-128 repressed gliomagenesis. We identified miR-128 as a glioma tumor suppressor that targets RTK signaling to repress giNSC self-renewal and enhance differentiation.
Collective and single cell behavior in epithelial contact inhibition.
Puliafito, A., Hufnagel, L., Neveu, P., Streichan, S., Sigal, A., Fygenson, D.K. & Shraiman, B.I.
Proc Natl Acad Sci U S A. 2012 Jan 17;109(3):739-44. doi:10.1073/pnas.1007809109. Epub 2012 Jan 6.
Control of cell proliferation is a fundamental aspect of tissue physiology central to morphogenesis, wound healing, and cancer. Although many of the molecular genetic factors are now known, the system level regulation of growth is still poorly understood. A simple form of inhibition of cell proliferation is encountered in vitro in normally differentiating epithelial cell cultures and is known as "contact inhibition." The study presented here provides a quantitative characterization of contact inhibition dynamics on tissue-wide and single cell levels. Using long-term tracking of cultured Madin-Darby canine kidney cells we demonstrate that inhibition of cell division in a confluent monolayer follows inhibition of cell motility and sets in when mechanical constraint on local expansion causes divisions to reduce cell area. We quantify cell motility and cell cycle statistics in the low density confluent regime and their change across the transition to epithelial morphology which occurs with increasing cell density. We then study the dynamics of cell area distribution arising through reductive division, determine the average mitotic rate as a function of cell size, and demonstrate that complete arrest of mitosis occurs when cell area falls below a critical value. We also present a simple computational model of growth mechanics which captures all aspects of the observed behavior. Our measurements and analysis show that contact inhibition is a consequence of mechanical interaction and constraint rather than interfacial contact alone, and define quantitative phenotypes that can guide future studies of molecular mechanisms underlying contact inhibition.
NMDA mediated contextual conditioning changes miRNA expression.
Kye, M.J., Neveu, P., Lee, Y.S., Zhou, M., Steen, J.A., Sahin, M., Kosik, K.S. & Silva, A.J.
PLoS One. 2011;6(9):e24682. doi: 10.1371/journal.pone.0024682. Epub 2011 Sep 12.
We measured the expression of 187 miRNAs using quantitative real time PCR in the hippocampal CA1 region of contextually conditioned mice and cultured embryonic rat hippocampal neurons after neuronal stimulation with either NMDA or bicuculline. Many of the changes in miRNA expression after these three types of stimulation were similar. Surprisingly, the expression level of half of the 187 measured miRNAs was changed in response to contextual conditioning in an NMDA receptor-dependent manner. Genes that control miRNA biogenesis and components of the RISC also exhibited activity induced expression changes and are likely to contribute to the widespread changes in the miRNA profile. The widespread changes in miRNA expression are consistent with the finding that genes up-regulated by contextual conditioning have longer 3' UTRs and more predicted binding sites for miRNAs. Among the miRNAs that changed their expression after contextual conditioning, several inhibit inhibitors of the mTOR pathway. These findings point to a role for miRNAs in learning and memory that includes mTOR-dependent modulation of protein synthesis.
The miRNA system: Bifurcation Points of Cancer and Neurodegeneration.
Kosik, K.S., Neveu, P. & Banerjee, S.
in: 'Two Faces of Evil: Cancer and Neurodegeneration', 2011 (Eds: T. Curran, Y. Christen), Springer, Heidelberg
Developmental programs direct cells toward specific fates by harnessing epigenetic mechanisms to progressively limit their potential and canalize changes in cell state toward a specialized terminal identity. Controlling proliferation on the one hand, and apoptosis on the other, are among the paramount twin hazards faced by the cell over its life span. Loosening of controls over proliferation clearly leads to cancer and loosening of controls over apoptosis may lead to degeneration. The genetic logic circuits that regulate proliferation and apoptosis lie at the core of cellular function, and it follows from the complexity of this circuitry that many different pathways exist in nearly all cells that can lead to cancer or degeneration. In neurons, microRNAs (miRNAs) have assumed a specialized role in regulating synaptic plasticity locally at synapses. This emerging role for miRNAs introduces another candidate pathway in the exploration of the underlying molecular mechanisms of neurodegeneration.
MicroRNA profiling reveals two distinct p53-related human pluripotent stem cell states.
Neveu, P., Kye, M.J., Qi, S., Buchholz, D.E., Clegg, D.O., Sahin, M., Park, I.H., Kim, K.S., Daley, G.Q., Kornblum, H.I., Shraiman, B.I. & Kosik, K.S.
Cell Stem Cell. 2010 Dec 3;7(6):671-81.
Reprogramming methodologies have provided multiple routes for achieving pluripotency. However, pluripotency is generally considered to be an almost singular state, with subtle differences described between induced pluripotent stem cells (iPSCs) and embryonic stem cells (ESCs). We profiled miRNA expression levels across 49 human cell lines, including ESCs, iPSCs, differentiated cells, and cancer cell lines. We found that the resulting miRNA profiles divided the iPSCs and hESCs examined into two distinct categories irrespective of the cell line origin. The miRNAs that defined these two pluripotency categories also distinguished cancer cells from differentiated cells. Transcriptome analysis suggested that several gene sets related to p53 distinguished these categories, and overexpression of the p53-targeting miRNAs miR-92 and miR-141 in iPSCs was sufficient to change their classification status. Thus, our results suggest a subdivision of pluripotent stem cell states that is independent of their origin but related to p53 network status.
MicroRNA regulation of neural stem cells and neurogenesis.
Shi, Y., Zhao, X., Hsieh, J., Wichterle, H., Impey, S., Banerjee, S., Neveu, P. & Kosik, K.S.
J Neurosci. 2010 Nov 10;30(45):14931-6.
MicroRNAs are a class of small RNA regulators that are involved in numerous cellular processes, including development, proliferation, differentiation, and plasticity. The emerging concept is that microRNAs play a central role in controlling the balance between stem cell self-renewal and fate determination by regulating the expression of stem cell regulators. This review will highlight recent advances in the regulation of neural stem cell self-renewal and neurogenesis by microRNAs. It will cover microRNA functions during the entire process of neurogenesis, from neural stem cell self-renewal and fate determination to neuronal maturation, synaptic formation, and plasticity. The interplay between microRNAs and both cell-intrinsic and -extrinsic stem cell players, including transcription factors, epigenetic regulators, and ex
Photoactivation of the CreER T2 recombinase for conditional site-specific recombination with high spatiotemporal resolution.
Sinha, D.K., Neveu, P., Gagey, N., Aujard, I., Le Saux, T., Rampon, C., Gauron, C., Kawakami, K., Leucht, C., Bally-Cuif, L., Volovitch, M., Bensimon, D., Jullien, L. & Vriz, S.
Zebrafish. 2010 Jun;7(2):199-204.
We implemented a noninvasive optical method for the fast control of Cre recombinase in single cells of a live zebrafish embryo. Optical uncaging of the caged precursor of a nonendogeneous steroid by one- or two-photon illumination was used to restore Cre activity of the CreER(T2) fusion protein in specific target cells. This method labels single cells irreversibly by inducing recombination in an appropriate reporter transgenic animal and thereby can achieve high spatiotemporal resolution in the control of gene expression. This technique could be used more generally to investigate important physiological processes (e.g., in embryogenesis, organ regeneration, or carcinogenesis) with high spatiotemporal resolution (single cell and 10-min scales).
Photocontrol of protein activity in cultured cells and zebrafish with one- and two-photon illumination.
Sinha, D.K., Neveu, P., Gagey, N., Aujard, I., Benbrahim-Bouzidi, C., Le Saux, T., Rampon, C., Gauron, C., Goetz, B., Dubruille, S., Baaden, M., Volovitch, M., Bensimon, D., Vriz, S. & Jullien, L.
Chembiochem. 2010 Mar 22;11(5):653-63.
We have implemented a noninvasive optical method for the fast control of protein activity in a live zebrafish embryo. It relies on releasing a protein fused to a modified estrogen receptor ligand binding domain from its complex with cytoplasmic chaperones, upon the local photoactivation of a nonendogenous caged inducer. Molecular dynamics simulations were used to design cyclofen-OH, a photochemically stable inducer of the receptor specific for 4-hydroxy-tamoxifen (ER(T2)). Cyclofen-OH was easily synthesized in two steps with good yields. At submicromolar concentrations, it activates proteins fused to the ER(T2) receptor. This was shown in cultured cells and in zebrafish embryos through emission properties and subcellular localization of properly engineered fluorescent proteins. Cyclofen-OH was successfully caged with various photolabile protecting groups. One particular caged compound was efficient in photoinducing the nuclear translocation of fluorescent proteins either globally (with 365 nm UV illumination) or locally (with a focused UV laser or with two-photon illumination at 750 nm). The present method for photocontrol of protein activity could be used more generally to investigate important physiological processes (e.g., in embryogenesis, organ regeneration and carcinogenesis) with high spatiotemporal resolution.
Vulnerabilities in the tau network and the role of ultrasensitive points in tau pathophysiology.
Yuraszeck, T.M., Neveu, P., Rodriguez-Fernandez, M., Robinson, A., Kosik, K.S. & Doyle FJ, 3rd
PLoS Comput Biol. 2010 Nov 11;6(11):e1000997.
The multifactorial nature of disease motivates the use of systems-level analyses to understand their pathology. We used a systems biology approach to study tau aggregation, one of the hallmark features of Alzheimer's disease. A mathematical model was constructed to capture the current state of knowledge concerning tau's behavior and interactions in cells. The model was implemented in silico in the form of ordinary differential equations. The identifiability of the model was assessed and parameters were estimated to generate two cellular states: a population of solutions that corresponds to normal tau homeostasis and a population of solutions that displays aggregation-prone behavior. The model of normal tau homeostasis was robust to perturbations, and disturbances in multiple processes were required to achieve an aggregation-prone state. The aggregation-prone state was ultrasensitive to perturbations in diverse subsets of networks. Tau aggregation requires that multiple cellular parameters are set coordinately to a set of values that drive pathological assembly of tau. This model provides a foundation on which to build and increase our understanding of the series of events that lead to tau aggregation and may ultimately be used to identify critical intervention points that can direct the cell away from tau aggregation to aid in the treatment of tau-mediated (or related) aggregation diseases including Alzheimer's.
Single cell physiology.
Neveu P, Sinha D, Kettunen P, Vriz S, Jullien L, Bensimon D.
in 'Single Molecule Spectroscopy in Chemistry, Physics and Biology', 2009 (Eds.: A. Gräslund, R. Rigler, J. Widengren), Springer, Heidelberg, pp 305-16.
The possibility to control at specific times and specific places the activity of biomolecules (enzymes, transcription factors, RNA, hormones, etc.) is opening up new opportunities in the study of physiological processes at the single cell level in a live organism. Most existing gene expression systems allow for tissue specific induction upon feeding the organism with exogenous inducers (e.g., tetracycline). Local genetic control has earlier been achieved by micro-injection of the relevant inducer/repressor molecule, but this is an invasive and possibly traumatic technique. In this chapter, we present the requirements for a noninvasive optical control of the activity of biomolecules and review the recent advances in this new field of research.
A coordinated local translational control point at the synapse involving relief from silencing and MOV10 degradation.
Banerjee, S., Neveu, P. & Kosik, K.S.
Neuron. 2009 Dec 24;64(6):871-84.
Persistent changes in synaptic strength are locally regulated by both protein degradation and synthesis; however, the coordination of these opposing limbs is poorly understood. Here, we found that the RISC protein MOV10 was present at synapses and was rapidly degraded by the proteasome in an NMDA-receptor-mediated activity-dependent manner. We designed a translational trap to capture those mRNAs whose spatiotemporal translation is regulated by MOV10. When MOV10 was suppressed, a set of mRNAs--including alpha-CaMKII, Limk1, and the depalmitoylating enzyme lysophospholipase1 (Lypla1)--selectively entered the polysome compartment. We also observed that Lypla1 mRNA is associated with the brain-enriched microRNA miR-138. Using a photoconvertible translation reporter, Kaede, we analyzed the activity-dependent protein synthesis driven by Lypla1 and alpha-CaMKII 3'UTRs. We established this protein synthesis to be MOV10 and proteasome dependent. These results suggest a unifying picture of a local translational regulatory mechanism during synaptic plasticity.
Small and stable peptidic PEGylated quantum dots to target polyhistidine-tagged proteins with controlled stoichiometry.
Dif, A., Boulmedais, F., Pinot, M., Roullier, V., Baudy-Floc'h, M., Coquelle, F.M., Clarke, S., Neveu, P., Vignaux, F., Le Borgne, R., Dahan, M., Gueroui, Z. & Marchi-Artzner, V.
J Am Chem Soc. 2009 Oct 21;131(41):14738-46.
The use of the semiconductor quantum dots (QD) as biolabels for both ensemble and single-molecule tracking requires the development of simple and versatile methods to target individual proteins in a controlled manner, ideally in living cells. To address this challenge, we have prepared small and stable QDs (QD-ND) using a surface coating based on a peptide sequence containing a tricysteine, poly(ethylene glycol) (PEG), and an aspartic acid ligand. These QDs, with a hydrodynamic diameter of 9 +/- 1.5 nm, can selectively bind to polyhistidine-tagged (histag) proteins in vitro or in living cells. We show that the small and monodisperse size of QD-ND allows for the formation of QD-ND/histag protein complexes of well-defined stoichiometry and that the 1:1 QD/protein complex can be isolated and purified by gel electrophoresis without any destabilization in the nanomolar concentration range. We also demonstrate that QD-ND can be used to specifically label a membrane receptor with an extracellular histag expressed in living HeLa cells. Here, cytotoxicity tests reveal that cell viability remains high under the conditions required for cellular labeling with QD-ND. Finally, we apply QD-ND complexed with histag end binding protein-1 (EB1), a microtubule associated protein, to single-molecule tracking in Xenopus extracts. Specific colocalization of QD-ND/EB1 with microtubules during the mitotic spindle formation demonstrates that QD-ND and our labeling strategy provide an efficient approach to monitor the dynamic behavior of proteins involved in complex biological functions.
The cochaperone BAG2 sweeps paired helical filament- insoluble tau from the microtubule.
Carrettiero, D.C., Hernandez, I., Neveu, P., Papagiannakopoulos, T. & Kosik, K.S.
J Neurosci. 2009 Feb 18;29(7):2151-61.
Tau inclusions are a prominent feature of many neurodegenerative diseases including Alzheimer's disease. Their accumulation in neurons as ubiquitinated filaments suggests a failure in the degradation limb of the Tau pathway. The components of a Tau protein triage system consisting of CHIP/Hsp70 and other chaperones have begun to emerge. However, the site of triage and the master regulatory elements are unknown. Here, we report an elegant mechanism of Tau degradation involving the cochaperone BAG2. The BAG2/Hsp70 complex is tethered to the microtubule and this complex can capture and deliver Tau to the proteasome for ubiquitin-independent degradation. This complex preferentially degrades Sarkosyl insoluble Tau and phosphorylated Tau. BAG2 levels in cells are under the physiological control of the microRNA miR-128a, which can tune paired helical filament Tau levels in neurons. Thus, we propose that ubiquitinated Tau inclusions arise due to shunting of Tau degradation toward a less efficient ubiquitin-dependent pathway.
A Preliminary Mathematical Model to Investigate the Triggers Leading to Aggregation of Tau Protein in Alzheimer's Patients and Identify Intervention Points to Prevent Aggregation.
Yuraszeck T, Neveu P, Rodriguez M, Kosik KS, Robinson AS, Doyle FJ III.
Proc. FOSBE Conference, 2009.
Alcohol uncaging with fluorescence reporting: evaluation of o-acetoxyphenyl methyloxazolone precursors.
Gagey, N., Emond, M., Neveu, P., Benbrahim, C., Goetz, B., Aujard, I., Baudin, J.B. & Jullien, L.
Org Lett. 2008 Jun 19;10(12):2341-4. Epub 2008 May 27.
This paper evaluates a series of photolabile protecting groups with built-in fluorescence reporting. They rely on readily available o-acetoxyphenyl methyloxazolones as activated precursors. Alcohol substrates are easily caged. The resulting photoactivable esters exhibit large one- and two-photon uncaging cross sections. The alcohol substrates are quantitatively released in a 1:1 molar ratio with a strongly fluorescent coumarin coproduct that serves as a reporter to quantify substrate delivery.
A caged retinoic acid for one- and two-photon excitation in zebrafish embryos.
Neveu, P., Aujard, I., Benbrahim, C., Le Saux, T., Allemand, J.F., Vriz, S., Bensimon, D. & Jullien, L.
Angew Chem Int Ed Engl. 2008;47(20):3744-6. Europe PMC
Two-photon uncaging with fluorescence reporting: evaluation of the o-hydroxycinnamic platform.
Gagey, N., Neveu, P., Benbrahim, C., Goetz, B., Aujard, I., Baudin, J.B. & Jullien, L.
J Am Chem Soc. 2007 Aug 15;129(32):9986-98. Epub 2007 Jul 21.
This paper evaluates the o-hydroxycinnamic platform for designing efficient caging groups with fluorescence reporting upon one- and two-photon excitation. The model cinnamates are easily prepared in one step by coupling commercial or readily available synthons. They exhibit a large one-photon absorption that can be tuned in the near-UV range. Uncaging after one-photon excitation was investigated by 1H NMR, UV-vis absorption, and steady-state fluorescence emission. In the whole investigated series, the caged substrate is quantitatively released upon photolysis. At the same time, uncaging releases a strongly fluorescent coproduct that can be used as a reporter for quantitative substrate delivery. The quantum yield of double bond photoisomerization leading to uncaging after one-photon absorption mostly lies in the 10% range. Taking advantage of the favorable photophysical properties of the uncaging coproduct, we use a series of techniques based on fluorescence emission to measure the action uncaging cross sections with two-photon excitation of the present cinnamates. Exhibiting values in the 1-10 GM range at 750 nm, they satisfactorily compare with the most efficient caging groups reported to date. Noticeably, the uncaging behavior with two-photon excitation is retained in vivo as suggested by the results observed in living zebrafish embryos. Reliable structure property relationships were extracted from analysis of the present collected data. In particular, the careful kinetic analysis allows us to discuss the relevance of the o-hydroxycinnamic platform for diverse caging applications with one- and two-photon excitation.
Two-photon uncaging with the efficient 3,5-dibromo-2,4-dihydroxycinnamic caging group.
Gagey, N., Neveu, P. & Jullien, L.
Angew Chem Int Ed Engl. 2007;46(14):2467-9. Europe PMC
o-nitrobenzyl photolabile protecting groups with red-shifted absorption: syntheses and uncaging cross-sections for one- and two-photon excitation.
Aujard, I., Benbrahim, C., Gouget, M., Ruel, O., Baudin, J.B., Neveu, P. & Jullien, L.
Chemistry. 2006 Sep 6;12(26):6865-79.
We evaluated the o-nitrobenzyl platform for designing photolabile protecting groups with red-shifted absorption that could be photolyzed upon one- and two-photon excitation. Several synthetic pathways to build different conjugated o-nitrobenzyl backbones, as well as to vary the benzylic position, are reported. Relative to the reference 4,5-dimethoxy-2-nitrobenzyl group, several o-nitrobenzyl derivatives exhibit a large and red-shifted one-photon absorption within the near-UV range. Uncaging after one-photon excitation was studied by measuring UV-visible absorption and steady-state fluorescence emission on model caged ethers and esters. In the whole series investigated, the caged substrates were released cleanly upon photolysis. Quantum yields of uncaging after one-photon absorption lie within the 0.1-1 % range. We observed that these drop as the maximum wavelength absorption of the o-nitrobenzyl protecting group is increased. A new method based on fluorescence correlation spectroscopy (FCS) after two-photon excitation was used to measure the action uncaging cross section for two-photon excitation. The series of o-nitrobenzyl caged fluorescent coumarins investigated exhibit values within the 0.1-0.01 Goeppert-Mayer (GM) range. Such results are in line with the low quantum yields of uncaging associated with cross-sections of 1-50 GM for two-photon absorption. Although the cross-sections for one- and two-photon absorption of o-nitrobenzyl photolabile protecting groups can be readily improved, we emphasize the difficulty in enlarging the corresponding action uncaging cross-sections in view of the observed trend of their quantum yield of uncaging.