This document summarizes fluorescent biosensors for intracellular signaling molecules. It describes various types of biosensors including GFP-based sensors that change brightness or use FRET to report on analytes like pH, calcium, cyclic nucleotides. Translocation sensors are described that detect protein movement from cytosol to membranes or nuclei in response to signals. Photoconvertible proteins like Dendra are presented as tools to visualize protein degradation. The mechanisms and applications of FRET-based, single fluorescent protein, and circularly permuted fluorescent protein sensors are outlined. Examples demonstrate calcium, kinase, and redox signaling biosensors. Imaging with HyPer revealed hydrogen peroxide production occurs locally near receptor tyrosine kinases and does not diffuse far within cells. A dual
Signal transduction in plant defense responsesVINOD BARPA
Signal transduction a Process by which a cell converts one kind of signal into another. Plant disease resistance and susceptibility are gov¬erned by the combined genotypes of host and pathogen and depend on a complex exchange of signals and responses occurring under given environmental con¬ditions. During the long process of host-pathogen co-evolution, plants have developed various elaborate mechanisms to ward off pathogen attack. Whereas some of these defense mechanisms are preformed and provide physical and chemical barriers to hinder pathogen infection, others are induced only after pa¬thogen attack. Similar to animal immune responses, induced plant defense responses involve a network of signal transduction and the rapid activation of gene expression following pathogen infection. They do not have immune system and locomotary organs to escape environmental challenges and biotic stresses. In plant, nature has provided them some preformed and inducible defense resistance. Host recognition of invading pathogen is often determined by the so called “gene for gene” interaction between avirulence (avr) gene of pathogen and corresponding resistance (R) gene of host (Flor, 1971) which encode receptor for the recognition of specific elicitor or ligand encoded directly or indirectly by pathogen avr gene. Recent studies have revealed intriguing parallels between animal and plant defense responses as demonstrated by the structural and functional conservation of some of their signal transduction processes. Furthermore, signaling components such as G proteins, NADPH oxidase, H202, salicylic acid (SA, and aspirin), mitogen-activated protein kinases (MAPK), and transcription factors have been shown to be associated with or participate in both animal and plant defense responses, suggesting the presence of con¬served signaling pathways for host defenses in diverse higher eukaryotes.
REGULATION OF
GENE EXPRESSION
IN PROKARYOTES & EUKARYOTES .
This presentation is enriched with lots of information of gene expression with many pictures so that anyone can understand gene expression easily.
Gene expression is the process by which the information encoded in a gene is used to direct the assembly of a protein molecule.
Gene expression is explored through a study of protein structure and function, transcription and translation, differentiation and stem cells.
It is the process by which information from a gene is used in the synthesis of a functional gene product.
These products are often proteins, but in non-protein coding genes such as ribosomal RNA (rRNA), transfer RNA (tRNA) or small nuclear RNA (snRNA) genes, the product is a functional RNA.
The process of gene expression is used by all known life - eukaryotes (including multicellular organisms), prokaryotes (bacteria and archaea)
Regulation of gene expression:
Regulation of gene expression includes a wide range of mechanisms that are used by cells to increase or decrease the production of specific gene products (protein or RNA).
Gene regulation is essential for viruses, prokaryotes and eukaryotes as it increases the versatility and adaptability of an organism by allowing the cell to express protein when needed.
CLASSIFICATION OF GENE WITH RESPECT TO THEIR EXPRESSION:
Constitutive ( house keeping) genes:
Are expressed at a fixed rate, irrespective to the cell condition.
Their structure is simpler.
Controllable genes:
Are expressed only as needed. Their amount may increase or decrease with respect to their basal level in different condition.
Their structure is relatively complicated with some response elements.
TYPES OF REGULATION OF GENE:
positive & negative regulation.
Steps involving gene regulation of prokaryotes & eukaryotes.
Operon-structure,classification of mechanisms- lac operon,tryptophan operon ,
and many things related to gene expression.
This is a video slide so anyone can understand this topic easily by seeing pictures included in this slide.
Signal transduction in plant defense responsesVINOD BARPA
Signal transduction a Process by which a cell converts one kind of signal into another. Plant disease resistance and susceptibility are gov¬erned by the combined genotypes of host and pathogen and depend on a complex exchange of signals and responses occurring under given environmental con¬ditions. During the long process of host-pathogen co-evolution, plants have developed various elaborate mechanisms to ward off pathogen attack. Whereas some of these defense mechanisms are preformed and provide physical and chemical barriers to hinder pathogen infection, others are induced only after pa¬thogen attack. Similar to animal immune responses, induced plant defense responses involve a network of signal transduction and the rapid activation of gene expression following pathogen infection. They do not have immune system and locomotary organs to escape environmental challenges and biotic stresses. In plant, nature has provided them some preformed and inducible defense resistance. Host recognition of invading pathogen is often determined by the so called “gene for gene” interaction between avirulence (avr) gene of pathogen and corresponding resistance (R) gene of host (Flor, 1971) which encode receptor for the recognition of specific elicitor or ligand encoded directly or indirectly by pathogen avr gene. Recent studies have revealed intriguing parallels between animal and plant defense responses as demonstrated by the structural and functional conservation of some of their signal transduction processes. Furthermore, signaling components such as G proteins, NADPH oxidase, H202, salicylic acid (SA, and aspirin), mitogen-activated protein kinases (MAPK), and transcription factors have been shown to be associated with or participate in both animal and plant defense responses, suggesting the presence of con¬served signaling pathways for host defenses in diverse higher eukaryotes.
REGULATION OF
GENE EXPRESSION
IN PROKARYOTES & EUKARYOTES .
This presentation is enriched with lots of information of gene expression with many pictures so that anyone can understand gene expression easily.
Gene expression is the process by which the information encoded in a gene is used to direct the assembly of a protein molecule.
Gene expression is explored through a study of protein structure and function, transcription and translation, differentiation and stem cells.
It is the process by which information from a gene is used in the synthesis of a functional gene product.
These products are often proteins, but in non-protein coding genes such as ribosomal RNA (rRNA), transfer RNA (tRNA) or small nuclear RNA (snRNA) genes, the product is a functional RNA.
The process of gene expression is used by all known life - eukaryotes (including multicellular organisms), prokaryotes (bacteria and archaea)
Regulation of gene expression:
Regulation of gene expression includes a wide range of mechanisms that are used by cells to increase or decrease the production of specific gene products (protein or RNA).
Gene regulation is essential for viruses, prokaryotes and eukaryotes as it increases the versatility and adaptability of an organism by allowing the cell to express protein when needed.
CLASSIFICATION OF GENE WITH RESPECT TO THEIR EXPRESSION:
Constitutive ( house keeping) genes:
Are expressed at a fixed rate, irrespective to the cell condition.
Their structure is simpler.
Controllable genes:
Are expressed only as needed. Their amount may increase or decrease with respect to their basal level in different condition.
Their structure is relatively complicated with some response elements.
TYPES OF REGULATION OF GENE:
positive & negative regulation.
Steps involving gene regulation of prokaryotes & eukaryotes.
Operon-structure,classification of mechanisms- lac operon,tryptophan operon ,
and many things related to gene expression.
This is a video slide so anyone can understand this topic easily by seeing pictures included in this slide.
1. Levels of gene regulation
The observation that differences in the RNA and protein content of different tissues are not paralleled by significant differences in their DNA content indicates that the process whereby DNA produces mRNA must be the level at which gene expression is regulated in eukaryotes. In bacteria this process involves only a single stage, that of transcription, in which RNA copy of the DNA is produced by the enzyme RNA polymerase. Even while this process is still occurring, ribosomes attach to the nascent RNA chain and begin to translate it into protein. Hence cases
of gene regulation in bacteria, such as the switching on of the synthesis of the enzyme β-galactosidase in response to the presence of lactose (its substrate), are mediated by increased transcription of the appropriate gene. Clearly, a similar regulation of gene transcription in different tissues, or in response to substances such as steroid hormones which induce the synthesis of new proteins, represents an attractive method of gene regulation in eukaryotes.
In contrast to the situation in bacteria, however, a number of stages intervene between the initial synthesis of the primary RNA transcript and the eventual production of mRNA (Fig. 1).
The initial transcript is modified at its 5′ end by the addition of a cap structure containing a modified guanosine residue and is subsequently cleaved near its 3′ end, followed by the addition of up to 200 adenosine residues in a process known as polyadenylation. Subsequently, intervening sequences or introns, which interrupt the protein-coding sequence in both the DNA and the primary transcript of many genes. Although this produces a functional mRNA, the spliced molecule must then be transported from the nucleus, where these processes occur, to the cytoplasm where it can be translated into protein.
Basics of Undergraduate/university fellows
Transcription is more complicated in eukaryotes than in prokaryotes because
eukaryotes possess three different classes of RNA polymerases and because of the
way in which transcripts are processed to their functional forms.
More proteins and transcription factors are involved in eukaryotic transcription.
this is a presentation on gene expression vector that includes what is expression vector, how many types of expression vector and difference between cloning and expression vector
This PPT has described how to produce soluble anf high amount of recombinant protein in E.coli host. This PPT has mentioned different expression vectors, different E.coli Expression host strain and other strategies for getting high expression of desired gene.
cell signaling is part of any communication process that governs basic activities of cells and coordinates multiple-cell actions. The ability of cells to perceive and correctly respond to their microenvironment is the basis of development, tissue repair, and immunity, as well as normal tissue homeostasis
1. Levels of gene regulation
The observation that differences in the RNA and protein content of different tissues are not paralleled by significant differences in their DNA content indicates that the process whereby DNA produces mRNA must be the level at which gene expression is regulated in eukaryotes. In bacteria this process involves only a single stage, that of transcription, in which RNA copy of the DNA is produced by the enzyme RNA polymerase. Even while this process is still occurring, ribosomes attach to the nascent RNA chain and begin to translate it into protein. Hence cases
of gene regulation in bacteria, such as the switching on of the synthesis of the enzyme β-galactosidase in response to the presence of lactose (its substrate), are mediated by increased transcription of the appropriate gene. Clearly, a similar regulation of gene transcription in different tissues, or in response to substances such as steroid hormones which induce the synthesis of new proteins, represents an attractive method of gene regulation in eukaryotes.
In contrast to the situation in bacteria, however, a number of stages intervene between the initial synthesis of the primary RNA transcript and the eventual production of mRNA (Fig. 1).
The initial transcript is modified at its 5′ end by the addition of a cap structure containing a modified guanosine residue and is subsequently cleaved near its 3′ end, followed by the addition of up to 200 adenosine residues in a process known as polyadenylation. Subsequently, intervening sequences or introns, which interrupt the protein-coding sequence in both the DNA and the primary transcript of many genes. Although this produces a functional mRNA, the spliced molecule must then be transported from the nucleus, where these processes occur, to the cytoplasm where it can be translated into protein.
Basics of Undergraduate/university fellows
Transcription is more complicated in eukaryotes than in prokaryotes because
eukaryotes possess three different classes of RNA polymerases and because of the
way in which transcripts are processed to their functional forms.
More proteins and transcription factors are involved in eukaryotic transcription.
this is a presentation on gene expression vector that includes what is expression vector, how many types of expression vector and difference between cloning and expression vector
This PPT has described how to produce soluble anf high amount of recombinant protein in E.coli host. This PPT has mentioned different expression vectors, different E.coli Expression host strain and other strategies for getting high expression of desired gene.
cell signaling is part of any communication process that governs basic activities of cells and coordinates multiple-cell actions. The ability of cells to perceive and correctly respond to their microenvironment is the basis of development, tissue repair, and immunity, as well as normal tissue homeostasis
Scotmas chlorine dioxide water treatment systems are used extensively in the poultry industry to improve health, welfare and overall production efficiency by reducing mortality and increasing feed conversion. Learn more about our unique ClO2 technology.
Signal transducing machinery as targets for potential drugs.
Drugs:-
a). Diclofenac- for treating cholera toxin
b). Fasentin- for treating insulin signalling
Generation of MRP2 Efflux Transporter Knock-Out in HepaRG Cell Linemdmitc
MilliporeSigma's Jennifer Pratt recently presented a poster at the 2016 AAPS/ITC Transporter Workshop demonstrating the utility of HepaRG MRP2 Knockout cells for investigating drug-transporter interactions in the liver involving MRP2.
KDM5 epigenetic modifiers as a focus for drug discoveryChristopher Wynder
A summary presentation of my scientific work.
My laboratory focused on an enzyme KDM5b (aka PLU-1, JARID1b) that was widely expressed during development and played a key role in progression of breast cancer through HER-2.
My lab focused on understanding the key biochemical activity of the enzyme through dissecting the proteomic and genomic interactors.
Our results were confirmed through the use of ES cells, adult stem cells and mouse models.
Much of this work remains unpublished, please contact me for more information and/or access to any reagents that I still have as part of this work.
crwynder@gmail.com
Transcription factors of the nuclear factor κ B family are the paradigm for signaling dependent nuclear translocation and are ideally suited to analysis through image-based chemical genetic screening. The authors describe combining high-content image analysis with a compound screen to identify compounds affecting either nuclear import or export. Validation in silico and in vitro determined an EC50 for the nuclear export blocker leptomycin B of 2.4 ng/mL (4.4 nM). The method demonstrated high selectivity (Z′ >0.95), speed, and robustness in a screen of a compound collection. It identified the IκB protein kinase inhibitor BAY 11 7082 as an import inhibitor, the p38 mitogen-activated protein (MAP) kinase inhibitor PD98509 as an import enhancer, and phorbol ester as an export inhibitor. The results establish a robust method for identifying compounds regulating nucleocy- toplasmic import or export and also implicate MAP kinases in nuclear import of nuclear factor κ B
Neuro-symbolic is not enough, we need neuro-*semantic*Frank van Harmelen
Neuro-symbolic (NeSy) AI is on the rise. However, simply machine learning on just any symbolic structure is not sufficient to really harvest the gains of NeSy. These will only be gained when the symbolic structures have an actual semantics. I give an operational definition of semantics as “predictable inference”.
All of this illustrated with link prediction over knowledge graphs, but the argument is general.
Connector Corner: Automate dynamic content and events by pushing a buttonDianaGray10
Here is something new! In our next Connector Corner webinar, we will demonstrate how you can use a single workflow to:
Create a campaign using Mailchimp with merge tags/fields
Send an interactive Slack channel message (using buttons)
Have the message received by managers and peers along with a test email for review
But there’s more:
In a second workflow supporting the same use case, you’ll see:
Your campaign sent to target colleagues for approval
If the “Approve” button is clicked, a Jira/Zendesk ticket is created for the marketing design team
But—if the “Reject” button is pushed, colleagues will be alerted via Slack message
Join us to learn more about this new, human-in-the-loop capability, brought to you by Integration Service connectors.
And...
Speakers:
Akshay Agnihotri, Product Manager
Charlie Greenberg, Host
Slack (or Teams) Automation for Bonterra Impact Management (fka Social Soluti...Jeffrey Haguewood
Sidekick Solutions uses Bonterra Impact Management (fka Social Solutions Apricot) and automation solutions to integrate data for business workflows.
We believe integration and automation are essential to user experience and the promise of efficient work through technology. Automation is the critical ingredient to realizing that full vision. We develop integration products and services for Bonterra Case Management software to support the deployment of automations for a variety of use cases.
This video focuses on the notifications, alerts, and approval requests using Slack for Bonterra Impact Management. The solutions covered in this webinar can also be deployed for Microsoft Teams.
Interested in deploying notification automations for Bonterra Impact Management? Contact us at sales@sidekicksolutionsllc.com to discuss next steps.
Epistemic Interaction - tuning interfaces to provide information for AI supportAlan Dix
Paper presented at SYNERGY workshop at AVI 2024, Genoa, Italy. 3rd June 2024
https://alandix.com/academic/papers/synergy2024-epistemic/
As machine learning integrates deeper into human-computer interactions, the concept of epistemic interaction emerges, aiming to refine these interactions to enhance system adaptability. This approach encourages minor, intentional adjustments in user behaviour to enrich the data available for system learning. This paper introduces epistemic interaction within the context of human-system communication, illustrating how deliberate interaction design can improve system understanding and adaptation. Through concrete examples, we demonstrate the potential of epistemic interaction to significantly advance human-computer interaction by leveraging intuitive human communication strategies to inform system design and functionality, offering a novel pathway for enriching user-system engagements.
UiPath Test Automation using UiPath Test Suite series, part 4DianaGray10
Welcome to UiPath Test Automation using UiPath Test Suite series part 4. In this session, we will cover Test Manager overview along with SAP heatmap.
The UiPath Test Manager overview with SAP heatmap webinar offers a concise yet comprehensive exploration of the role of a Test Manager within SAP environments, coupled with the utilization of heatmaps for effective testing strategies.
Participants will gain insights into the responsibilities, challenges, and best practices associated with test management in SAP projects. Additionally, the webinar delves into the significance of heatmaps as a visual aid for identifying testing priorities, areas of risk, and resource allocation within SAP landscapes. Through this session, attendees can expect to enhance their understanding of test management principles while learning practical approaches to optimize testing processes in SAP environments using heatmap visualization techniques
What will you get from this session?
1. Insights into SAP testing best practices
2. Heatmap utilization for testing
3. Optimization of testing processes
4. Demo
Topics covered:
Execution from the test manager
Orchestrator execution result
Defect reporting
SAP heatmap example with demo
Speaker:
Deepak Rai, Automation Practice Lead, Boundaryless Group and UiPath MVP
State of ICS and IoT Cyber Threat Landscape Report 2024 previewPrayukth K V
The IoT and OT threat landscape report has been prepared by the Threat Research Team at Sectrio using data from Sectrio, cyber threat intelligence farming facilities spread across over 85 cities around the world. In addition, Sectrio also runs AI-based advanced threat and payload engagement facilities that serve as sinks to attract and engage sophisticated threat actors, and newer malware including new variants and latent threats that are at an earlier stage of development.
The latest edition of the OT/ICS and IoT security Threat Landscape Report 2024 also covers:
State of global ICS asset and network exposure
Sectoral targets and attacks as well as the cost of ransom
Global APT activity, AI usage, actor and tactic profiles, and implications
Rise in volumes of AI-powered cyberattacks
Major cyber events in 2024
Malware and malicious payload trends
Cyberattack types and targets
Vulnerability exploit attempts on CVEs
Attacks on counties – USA
Expansion of bot farms – how, where, and why
In-depth analysis of the cyber threat landscape across North America, South America, Europe, APAC, and the Middle East
Why are attacks on smart factories rising?
Cyber risk predictions
Axis of attacks – Europe
Systemic attacks in the Middle East
Download the full report from here:
https://sectrio.com/resources/ot-threat-landscape-reports/sectrio-releases-ot-ics-and-iot-security-threat-landscape-report-2024/
UiPath Test Automation using UiPath Test Suite series, part 3DianaGray10
Welcome to UiPath Test Automation using UiPath Test Suite series part 3. In this session, we will cover desktop automation along with UI automation.
Topics covered:
UI automation Introduction,
UI automation Sample
Desktop automation flow
Pradeep Chinnala, Senior Consultant Automation Developer @WonderBotz and UiPath MVP
Deepak Rai, Automation Practice Lead, Boundaryless Group and UiPath MVP
Transcript: Selling digital books in 2024: Insights from industry leaders - T...BookNet Canada
The publishing industry has been selling digital audiobooks and ebooks for over a decade and has found its groove. What’s changed? What has stayed the same? Where do we go from here? Join a group of leading sales peers from across the industry for a conversation about the lessons learned since the popularization of digital books, best practices, digital book supply chain management, and more.
Link to video recording: https://bnctechforum.ca/sessions/selling-digital-books-in-2024-insights-from-industry-leaders/
Presented by BookNet Canada on May 28, 2024, with support from the Department of Canadian Heritage.
DevOps and Testing slides at DASA ConnectKari Kakkonen
My and Rik Marselis slides at 30.5.2024 DASA Connect conference. We discuss about what is testing, then what is agile testing and finally what is Testing in DevOps. Finally we had lovely workshop with the participants trying to find out different ways to think about quality and testing in different parts of the DevOps infinity loop.
PHP Frameworks: I want to break free (IPC Berlin 2024)Ralf Eggert
In this presentation, we examine the challenges and limitations of relying too heavily on PHP frameworks in web development. We discuss the history of PHP and its frameworks to understand how this dependence has evolved. The focus will be on providing concrete tips and strategies to reduce reliance on these frameworks, based on real-world examples and practical considerations. The goal is to equip developers with the skills and knowledge to create more flexible and future-proof web applications. We'll explore the importance of maintaining autonomy in a rapidly changing tech landscape and how to make informed decisions in PHP development.
This talk is aimed at encouraging a more independent approach to using PHP frameworks, moving towards a more flexible and future-proof approach to PHP development.
"Impact of front-end architecture on development cost", Viktor TurskyiFwdays
I have heard many times that architecture is not important for the front-end. Also, many times I have seen how developers implement features on the front-end just following the standard rules for a framework and think that this is enough to successfully launch the project, and then the project fails. How to prevent this and what approach to choose? I have launched dozens of complex projects and during the talk we will analyze which approaches have worked for me and which have not.
The Art of the Pitch: WordPress Relationships and SalesLaura Byrne
Clients don’t know what they don’t know. What web solutions are right for them? How does WordPress come into the picture? How do you make sure you understand scope and timeline? What do you do if sometime changes?
All these questions and more will be explored as we talk about matching clients’ needs with what your agency offers without pulling teeth or pulling your hair out. Practical tips, and strategies for successful relationship building that leads to closing the deal.
5. Translocation Biosensors – Tools for Signaling
Pathway Analysis
Translocation of a proteins from the cytosol to receptor
complexes at the plasma membrane:
- kinase receptors activation
Akt3-GFP in untreated cells
(upper) and in activated cells
(lower).
Akt3 is a serine/threonine kinase
that plays a key in regulating cell
survival, insulin signaling,
angiogenesis and tumor formation.
6. Translocation Biosensors – Tools for Signaling
Pathway Analysis
Translocation of a proteins from cytosol to nucleus:
- transcription factor activation
- caspase activation (apoptose)
Translocation of HIF-1a – GFP fusion protein from the
cytosol (a) to the nucleus (b) in response to PDT.
Photodynamic therapy-mediated hypoxia-independent activation of the
hypoxia inducible factor 1a (HIF-1a) pathway.
7. Dendra - a monomeric mutant of greento-red photoconvertible FP from
Dendronephthya
Dendronephthya sp.
300
400
500
600
Wavelength, nm
Gurskaya et al. Nat. Biotech. 2006
700
8. Visualization of target protein degradation using
Dendra2 photoactivatable protein
Green fluorescence
intensity depends on
both protein synthesis
and degradation
Expression of
Dendra2-tagged protein
Dendra2 photoconversion
in whole cell
Time-lapse
Red fluorescence
intensity depends only
on protein degradation
t0
t1 ...
tn
Quantification
Red 1
fluorescence
0.5
1/2
Time
9. FRET-based biosensors
Molecular sensors (pH, Ca2+, cAMP, kinases, redox, H2O2, etc.) on the base of FRET or
brightness changes.
(a) Biosensors based on a ligand-dependent protein–protein interaction. Cameleons (based
on a fusion of calmodulin and M13) and GTPase biosensors (based on a fusion of the GTPase
and its effector) fall into this category. (b) Post-translational modification biosensor (i.e., for
a kinase). (c) Protease substrate-type biosensor. (d) Biosensor based on conformational
change of a single protein.
11. Genetically encoded fluorescent sensor of ERK activity
The MAPK family is a class of serine/threonine kinases that includes the ERK, p38, and JNK
subfamilies. Members of the ERK subfamily are essential for numerous, diverse physiological
functions, including cellular differentiation, proliferation and neuronal plasticity, and their
activities are up-regulated in many cancers.
Fluorescence lifetime images of HEK293 cells
transfected with EKARcyto before and after (12 min)
addition of EGF (100 ng/ml).
12. Tyrosine Phosphorylation of Cytoplasmic Domain of
EGFR Monitored by FRET
Lippincott-Schwartz, Snapp and Kenworthy Nat. Rev. Mol. Cell Biol. 2:444, 2001
13. Tyrosine Phosphorylation of Cytoplasmic Domain of
EGFR Monitored by FRET
Verveer et al., Science 290:1567, 2000
14. Single FP-based biosensors
Molecular sensors (pH, Ca2+, cAMP, kinases, redox, H2O2, etc.) on the base of FRET or
brightness changes.
(a) Single FP biosensor based on intrinsic (i.e., pH) sensitivity. (b) Single FP biosensor
based on the extrinsic sensitivity (i.e., Ca2+) of a genetically fused domain (i.e.,
calmodulin). (c) GCaMP X-ray crystal structure.45 Linker regions that were not visible
in the crystal structure are represented with dashed lines.
16. FP biosensor structure and imaging
Molecular sensors (pH, Ca2+, cAMP, kinases, redox, H2O2, etc.) on the base of FRET or
brightness changes.
0s
10 s
10.8 s
11.3 s
(A) GCaMP2, a calcium indicator
constructed with a circularly permutated
EGFP fused to calmodulin and the
calmodulin-binding domain of myosin
light chain kinase (M13 domain) in the
absence of calcium.
(B) GCamP2 structure when bound to
calcium.
(C–F) Widefield fluorescence calcium
imaging in the cytosol of HeLa cells
expressing a calcium biosensor. (C) Real
color image of two cells, t = 0,
histamine (10 M) added; (D)
pseudocolored ratio image of two HeLa
cells as a calcium wave initiates in the
upper cell, t = 10 s. (E–F) The calcium
wave propagates through the cytoplasm
of both cells.
23. Research questions:
-Which cellular compartment is responsible for H2O2
production in RTKs signaling?
-When H2O2 appears?
-What is the diffusion distance of H2O2 within the cell?
33. H2O2 production by HeLa-Kyoto cells
stimulated with EGF
Mishina et al, ARS 2011
34. H2O2 production by HeLa-Kyoto cells
stimulated with EGF
Scale 5 m
Mishina et al, ARS 2011
35. H2O2 production by HeLa-Kyoto cells
stimulated with EGF
-H2O2 production/Nox activity
co-localizes with activated RTK
-H2O2 does not diffuse for a
long distance
-Nox activity translocates from
the PM to the endosomes
Mishina et al, ARS 2011
42. H2O2 production at the ER surface imaged using
HyPer-TA
Mishina et al, ARS 2011
43. Single system controls H2O2 production in fibroblasts
Co-localizes with PTP-1B
phosphatase
Co-localizes with active
RTK
Mishina et al, ARS 2011
44. -Which cellular compartment is responsible for NADPH
oxidases activation and H2O2 production in RTKs signalling?
Epithelial cells – Endosomes, ER membrane;
Fibroblasts – Plasma membrane, ER membrane.
45. -What is diffusion distance of H2O2 within the cell?
Estimated to be ~1m or even less. However, may vary in
different cell types.