Final Report Summary - PI3K-C2-SIG (Signalling mechanisms of the class II isoforms of PI 3-kinase)
This Marie Curie Postdoctoral Fellowship (IIF) aimed to uncover the signalling mechanisms and functions of the class II isoforms of the PI 3-kinase family. The class II PI 3-kinases (further referred to as PI3K-C2) are the least studied amongst the family and very little was known about these isoforms at the start of this project. Over the past two years, the Fellow has (a) worked on unpublished knockin (KI) mouse lines with genetically inactivated PI3K-C2 isoforms and characterized a new conditional PI3K-C2a KI mouse line, (b) set up new methodology for the 3D/4D analysis of organelles and proteins, (c) discovered an exciting new role for PI3K-C2a in signal transduction, and (d) reinforced the collaborative academia network between Europe and Australia.
Description of the work and main results:
1. Coupling of specific class II PI3K isoforms to distinct classes of receptors
- New findings have necessitated a focus of the project on the PI3K-C2a isoform of PI3K: Homozygous PI3K-C2a KI mice are embryonic lethal (at day E7.5) in contrast to homozygous PI3K-C2b KI mice, which are viable and fertile. At the start of the project, the generation of mice with floxed PI3K-C2a alleles became available to me. The Fellow has independently worked toward (i) establishing the new mouse line PI3K-C2afl/fl at the Host Institute (first at QMUL, now at UCL) and for a collaborating laboratory in France, (ii) initiating the PI3K-C2afl/flxCreERT2 cross, and (iii) characterized a new strategy for conditionally inactivating PI3K-C2a in mice.
In (iii), the Fellow applied a method of inducible Cre-mediated recombination of the floxed PI3K-C2a alleles following retroviral introduction of Cre recombinase: MEFs were generated from E13.5 embryos (PI3K-C2awt/wt, PI3K-C2afl/wt, PI3K-C2afl/fl) and infected (4 days) with retrovirus carrying GFP-fused, tamoxifen-inducible Cre recombinase (GFP-CreERT2). High titer retrovirus was prepared by lipofection of confluent HEK293T cultures and harvested as supernatants. Infection of MEFs occurred within 3-5 days and checked by GFP fluorescence. Addition of tamoxifen successfully induced recombination at the genomic and mRNA levels after 24-72 hours. Whole lysates harvested for SDS-PAGE and western blotting showed expression of mutant PI3K-C2a from 24 hours and a full KI by 72 hours. These data show that this strategy is effective for conditionally inactivating PI3K-C2a, albeit with a reduction of the mutant protein to about 40%. We are now cross-breeding the PI3K-C2afl/fl x CreERT2 lines, and we have been able to recapitulate the finding in tamoxifen-induced MEFs from PI3K-C2afl/fl/CreERT2 embryos.
- New role for PI3K-C2a in signalling: In order to assess the impact of inactivating PI3K-C2a, the Fellow has tested a number of cell types including MEFs, keratinocytes, and macrophages. These cells were also derived from the heterozygous PI3K-C2a mouse line (PI3K-C2aD1268A/wt), which has been characterized in the Host Laboratory. Cells were subjected to a number of treatment conditions including serum starvation, growth factor readdition (to induce cilia formation in MEFs - later published by an Italian research group on March 2014 in Dev Cell), and stimulation of specific receptors in macrophages. The Applicant has discovered that signalling by specific ligands is affected in bone marrow-derived macrophages (BMMs) from PI3K-C2aD1268A/wt compared to wild-type littermates. Interestingly, neither transferrin-mediated endocytosis nor phagocytosis of IgG-coated beads (internalization assay) appeared to be affected in PI3K-C2aD1268A/wt cells. The project has since focussed on elucidating the role of PI3K-C2a and to delineate the molecular details on how this kinase works in the identified signalling pathway (objectives 2 and 3).
2. Lipid-/protein kinase-based signalling pathways controlled by class II PI3Ks
• Lipid profiling of 3-phosphoinositides in cells with inactivation of one or more PI3K-C2 isoforms. To answer the question: which 3-phosphoinositides are affected upon inactivation of PI3K-C2 isoforms.
Current work in this aspect was centered on PI3K-C2b owing to a manuscript developing on this isoform. We are currently carrying out similar experiments for PI3K-C2a – collaborations with INSERM (Toulouse, France - PI3P mass assay) and the Babraham Institute (Cambridge, UK - HPLC and PIP3 mass spectrometry), and possibly with groups in Australia (HPLC). The Fellow, alongside other postdocs from the Host Laboratory, has also been using commercially ELISA (Echelon) that has recently become available to measure phosphoinositides. These data are now awaiting to be further confirmed by mass assay/HPLC.
• Protein kinase-based signalling pathways
We have since reassessed the time frame and taken into consideration the novelty and impact of our new findings on PI3K-C2a in signalling. While collaborations with Dr Cutillas or Activiomics (now part of hVIVO Retroscreen Virology Group, UK) will continue in the background, the focus of work of the Fellow has since been on interrogating the signalling pathways that have been identified, by standard immunoblotting for a wide variety of signal transduction pathways.
3. Class II PI3Ks in membrane topology and trafficking of receptors
This aim has been divided into two parts:-
• Contribution to manuscript on PI3K-C2b in a metabolic context – under revision in ‘Cell Reports’
The Fellow has worked toward setting up different imaging/analysis methods for studying the internalization and trafficking of the insulin ligand/receptors in primary MEFs and hepatocytes (5 months). While some of these methods work in MEFs, imaging of insulin ligand/receptors in hepatocytes has been very challenging: (i) it has been difficult to transiently transfect fluorescently-tagged receptors and/or markers into primary hepatocytes for imaging, and (ii) imaging of the fluorescent ligand insulin-Fitc has proven tricky in primary hepatocytes and there has been no software appropriate for analyzing images or videos from experiments at the QMUL. Since our move to UCL (January 2014) we have access to these essential softwares (IMARIS, VOLOCITY) and more specialized microscopes. This manuscript is now under final revision in Cell Reports.
• A role for PI3K-C2a in signalling?
The move of the Host Laboratory to UCL has offered improved resources for exploitation by the Fellow to effectively drive her project. High-resolution confocal microscopy was used to analyze the impact of PI3K-C2a inactivation on subcellular signal transduction sites. CreERT2-infected MEFs from PI3K-C2a floxed KI embryos appear to develop enlarged EEA1-positive early endosomes (EEs) upon tamoxifen induction (24-72 hours). Such enlarged EEs from homozygous PI3K-C2a inactivation are reminiscent of interference with PI3P function either by drug treatment (e.g. wortmannin) or by sequestration of PI3P through over-expression of a PI3P-binding module.
Expected final results and their potential impact and use (including the socio-economic impact and the wider societal implications of the project so far):
Over the past two years, work from the Fellow has opened up new, exciting avenues to understanding the signalling and physiological function of the class II PI3Ks. It has shed novel light on an unexpected role for the PI3K-C2a isoform in signal transduction, with possible clinical implications. Findings generated from work within the time frame of the Fellowship and beyond will have profound implications and may provide new pathways towards ongoing drug development (new PI3K inhibitors and activators) particularly in the emerging areas of infectious diseases and cancer. Importantly, this Fellowship has reinforced collaborative ties between the Host Laboratory in UK and the academia communities in Australia.
Researcher training activities/transfer of knowledge activities/integration activities:
- The Fellow has always worked toward transferring skills and expertise in membrane trafficking and imaging techniques to the Host Institution. Since arriving at the UCL, the Fellow has proposed upgrades to existing IMARIS software, which has then been funded by the microscopy department and this is appreciated by existing and new IMARIS users UCL-wide.
- Mutually beneficial long-term collaborations between the Host and international institutions will be fostered. The Supervisor and Fellow have recently hosted visit by the Director of the Biotechnology Program from the University of Queensland, Australia, where the Fellow came from. Successful publication of a work previously initiated as an international collaboration between the Supervisor and Fellow (Low et al. 2014 Nature Communications). Both industrial and academic collaborations with companies/laboratories in Australia will be strengthened.
- The Supervisor is very supportive of the professional career development of the Fellow.
*Presentation at scientific meetings and other scientific events:
- PI3-Kinases in Immunity, Inflammation and Infection. Babraham Institute, Cambridge, UK – June 2014.
- 2014 meeting of the International Cytokine and Interferon Society, Melbourne, Australia – October 2014.
The Fellow was awarded the highly competitive Milstein Travel Award to attend this conference.
*Outreach activities:
The Fellow was involved as a volunteering scientist in the filming of the new television advertisement for the Cancer Research UK.
Highlight clearly significant results:
- First characterization/report of PI3K-C2a KI mouse models
The Fellow has independently worked toward (i) establishing the new mouse line PI3K-C2afl/fl at the Host Institute (first at QMUL, now at UCL) and for a collaborating laboratory in France, (ii) initiating the PI3K-C2afl/flxCreERT2 cross, and (iii) characterized a new strategy for conditionally inactivating PI3K-C2a in mice.
In (iii), the Fellow applied a method of inducible Cre-mediated recombination of the floxed PI3K-C2a alleles following retroviral introduction of Cre recombinase. Our data show that the strategy is effective for conditionally inactivating PI3K-C2a, albeit with a reduction of the mutant protein to about 40%. We are now cross-breeding the PI3K-C2afl/fl x CreERT2 lines, and we have been able to recapitulate the finding in tamoxifen-induced MEFs from PI3K-C2afl/fl/CreERT2 embryos.
- New role for PI3K-C2a in signal transduction
Our data excitingly point to a novel role for PI3K-C2a in a specific signal transduction pathway. In line with the functional profiles, data from the signalling analyses corroborated that PI3K-C2aD1268A/wt BMMs have specifically dampened specific intracellular pathways. This has also been corroborated by high-resolution 3D/4D imaging of subcellular vesicular structures in PI3K-C2aD1268A/wt mutant cells upon ligand stimulation.
- Findings generated from work within the time frame of the Fellowship and beyond will have profound implications and may provide new pathways towards ongoing drug development (new PI3K inhibitors and activators) to benefit the biotechnology sector. Importantly, this Fellowship has reinforced collaborative ties between the Host Laboratory in UK and the academia and industry communities in Australia.
Description of the work and main results:
1. Coupling of specific class II PI3K isoforms to distinct classes of receptors
- New findings have necessitated a focus of the project on the PI3K-C2a isoform of PI3K: Homozygous PI3K-C2a KI mice are embryonic lethal (at day E7.5) in contrast to homozygous PI3K-C2b KI mice, which are viable and fertile. At the start of the project, the generation of mice with floxed PI3K-C2a alleles became available to me. The Fellow has independently worked toward (i) establishing the new mouse line PI3K-C2afl/fl at the Host Institute (first at QMUL, now at UCL) and for a collaborating laboratory in France, (ii) initiating the PI3K-C2afl/flxCreERT2 cross, and (iii) characterized a new strategy for conditionally inactivating PI3K-C2a in mice.
In (iii), the Fellow applied a method of inducible Cre-mediated recombination of the floxed PI3K-C2a alleles following retroviral introduction of Cre recombinase: MEFs were generated from E13.5 embryos (PI3K-C2awt/wt, PI3K-C2afl/wt, PI3K-C2afl/fl) and infected (4 days) with retrovirus carrying GFP-fused, tamoxifen-inducible Cre recombinase (GFP-CreERT2). High titer retrovirus was prepared by lipofection of confluent HEK293T cultures and harvested as supernatants. Infection of MEFs occurred within 3-5 days and checked by GFP fluorescence. Addition of tamoxifen successfully induced recombination at the genomic and mRNA levels after 24-72 hours. Whole lysates harvested for SDS-PAGE and western blotting showed expression of mutant PI3K-C2a from 24 hours and a full KI by 72 hours. These data show that this strategy is effective for conditionally inactivating PI3K-C2a, albeit with a reduction of the mutant protein to about 40%. We are now cross-breeding the PI3K-C2afl/fl x CreERT2 lines, and we have been able to recapitulate the finding in tamoxifen-induced MEFs from PI3K-C2afl/fl/CreERT2 embryos.
- New role for PI3K-C2a in signalling: In order to assess the impact of inactivating PI3K-C2a, the Fellow has tested a number of cell types including MEFs, keratinocytes, and macrophages. These cells were also derived from the heterozygous PI3K-C2a mouse line (PI3K-C2aD1268A/wt), which has been characterized in the Host Laboratory. Cells were subjected to a number of treatment conditions including serum starvation, growth factor readdition (to induce cilia formation in MEFs - later published by an Italian research group on March 2014 in Dev Cell), and stimulation of specific receptors in macrophages. The Applicant has discovered that signalling by specific ligands is affected in bone marrow-derived macrophages (BMMs) from PI3K-C2aD1268A/wt compared to wild-type littermates. Interestingly, neither transferrin-mediated endocytosis nor phagocytosis of IgG-coated beads (internalization assay) appeared to be affected in PI3K-C2aD1268A/wt cells. The project has since focussed on elucidating the role of PI3K-C2a and to delineate the molecular details on how this kinase works in the identified signalling pathway (objectives 2 and 3).
2. Lipid-/protein kinase-based signalling pathways controlled by class II PI3Ks
• Lipid profiling of 3-phosphoinositides in cells with inactivation of one or more PI3K-C2 isoforms. To answer the question: which 3-phosphoinositides are affected upon inactivation of PI3K-C2 isoforms.
Current work in this aspect was centered on PI3K-C2b owing to a manuscript developing on this isoform. We are currently carrying out similar experiments for PI3K-C2a – collaborations with INSERM (Toulouse, France - PI3P mass assay) and the Babraham Institute (Cambridge, UK - HPLC and PIP3 mass spectrometry), and possibly with groups in Australia (HPLC). The Fellow, alongside other postdocs from the Host Laboratory, has also been using commercially ELISA (Echelon) that has recently become available to measure phosphoinositides. These data are now awaiting to be further confirmed by mass assay/HPLC.
• Protein kinase-based signalling pathways
We have since reassessed the time frame and taken into consideration the novelty and impact of our new findings on PI3K-C2a in signalling. While collaborations with Dr Cutillas or Activiomics (now part of hVIVO Retroscreen Virology Group, UK) will continue in the background, the focus of work of the Fellow has since been on interrogating the signalling pathways that have been identified, by standard immunoblotting for a wide variety of signal transduction pathways.
3. Class II PI3Ks in membrane topology and trafficking of receptors
This aim has been divided into two parts:-
• Contribution to manuscript on PI3K-C2b in a metabolic context – under revision in ‘Cell Reports’
The Fellow has worked toward setting up different imaging/analysis methods for studying the internalization and trafficking of the insulin ligand/receptors in primary MEFs and hepatocytes (5 months). While some of these methods work in MEFs, imaging of insulin ligand/receptors in hepatocytes has been very challenging: (i) it has been difficult to transiently transfect fluorescently-tagged receptors and/or markers into primary hepatocytes for imaging, and (ii) imaging of the fluorescent ligand insulin-Fitc has proven tricky in primary hepatocytes and there has been no software appropriate for analyzing images or videos from experiments at the QMUL. Since our move to UCL (January 2014) we have access to these essential softwares (IMARIS, VOLOCITY) and more specialized microscopes. This manuscript is now under final revision in Cell Reports.
• A role for PI3K-C2a in signalling?
The move of the Host Laboratory to UCL has offered improved resources for exploitation by the Fellow to effectively drive her project. High-resolution confocal microscopy was used to analyze the impact of PI3K-C2a inactivation on subcellular signal transduction sites. CreERT2-infected MEFs from PI3K-C2a floxed KI embryos appear to develop enlarged EEA1-positive early endosomes (EEs) upon tamoxifen induction (24-72 hours). Such enlarged EEs from homozygous PI3K-C2a inactivation are reminiscent of interference with PI3P function either by drug treatment (e.g. wortmannin) or by sequestration of PI3P through over-expression of a PI3P-binding module.
Expected final results and their potential impact and use (including the socio-economic impact and the wider societal implications of the project so far):
Over the past two years, work from the Fellow has opened up new, exciting avenues to understanding the signalling and physiological function of the class II PI3Ks. It has shed novel light on an unexpected role for the PI3K-C2a isoform in signal transduction, with possible clinical implications. Findings generated from work within the time frame of the Fellowship and beyond will have profound implications and may provide new pathways towards ongoing drug development (new PI3K inhibitors and activators) particularly in the emerging areas of infectious diseases and cancer. Importantly, this Fellowship has reinforced collaborative ties between the Host Laboratory in UK and the academia communities in Australia.
Researcher training activities/transfer of knowledge activities/integration activities:
- The Fellow has always worked toward transferring skills and expertise in membrane trafficking and imaging techniques to the Host Institution. Since arriving at the UCL, the Fellow has proposed upgrades to existing IMARIS software, which has then been funded by the microscopy department and this is appreciated by existing and new IMARIS users UCL-wide.
- Mutually beneficial long-term collaborations between the Host and international institutions will be fostered. The Supervisor and Fellow have recently hosted visit by the Director of the Biotechnology Program from the University of Queensland, Australia, where the Fellow came from. Successful publication of a work previously initiated as an international collaboration between the Supervisor and Fellow (Low et al. 2014 Nature Communications). Both industrial and academic collaborations with companies/laboratories in Australia will be strengthened.
- The Supervisor is very supportive of the professional career development of the Fellow.
*Presentation at scientific meetings and other scientific events:
- PI3-Kinases in Immunity, Inflammation and Infection. Babraham Institute, Cambridge, UK – June 2014.
- 2014 meeting of the International Cytokine and Interferon Society, Melbourne, Australia – October 2014.
The Fellow was awarded the highly competitive Milstein Travel Award to attend this conference.
*Outreach activities:
The Fellow was involved as a volunteering scientist in the filming of the new television advertisement for the Cancer Research UK.
Highlight clearly significant results:
- First characterization/report of PI3K-C2a KI mouse models
The Fellow has independently worked toward (i) establishing the new mouse line PI3K-C2afl/fl at the Host Institute (first at QMUL, now at UCL) and for a collaborating laboratory in France, (ii) initiating the PI3K-C2afl/flxCreERT2 cross, and (iii) characterized a new strategy for conditionally inactivating PI3K-C2a in mice.
In (iii), the Fellow applied a method of inducible Cre-mediated recombination of the floxed PI3K-C2a alleles following retroviral introduction of Cre recombinase. Our data show that the strategy is effective for conditionally inactivating PI3K-C2a, albeit with a reduction of the mutant protein to about 40%. We are now cross-breeding the PI3K-C2afl/fl x CreERT2 lines, and we have been able to recapitulate the finding in tamoxifen-induced MEFs from PI3K-C2afl/fl/CreERT2 embryos.
- New role for PI3K-C2a in signal transduction
Our data excitingly point to a novel role for PI3K-C2a in a specific signal transduction pathway. In line with the functional profiles, data from the signalling analyses corroborated that PI3K-C2aD1268A/wt BMMs have specifically dampened specific intracellular pathways. This has also been corroborated by high-resolution 3D/4D imaging of subcellular vesicular structures in PI3K-C2aD1268A/wt mutant cells upon ligand stimulation.
- Findings generated from work within the time frame of the Fellowship and beyond will have profound implications and may provide new pathways towards ongoing drug development (new PI3K inhibitors and activators) to benefit the biotechnology sector. Importantly, this Fellowship has reinforced collaborative ties between the Host Laboratory in UK and the academia and industry communities in Australia.