The document summarizes research on the polarized trafficking of neuronal proteins like syntaxins and potassium channels. It finds that syntaxin 3 mediates the trafficking of NgCAM to axons, while potassium channel Kir2.3 is clustered in dendritic spines through its interaction with PSD-95. Precise localization of proteins through trafficking mechanisms is important for neuronal polarity and function.
Will Jackson presented his Master's defense talk on identifying interactions between the transcription factor Lhx2 and other neocortical proteins. He used a yeast two-hybrid screen to identify interacting proteins, finding that Lhx2 interacts with the RNA helicase Ddx50 and exonuclease Eri3. Further experiments showed the LIM domain of Lhx2 is necessary for binding to Eri3. These interactions may regulate transcription and mRNA stability during neocortical development. Future work will aim to clarify the functional roles and pathways of Lhx2 with its interacting partners.
The document summarizes a thesis defense presentation on the role of focal adhesion kinase (FAK) in vascular smooth muscle cell (SMC) migration. The results showed that FAK mediates SMC migration toward platelet-derived growth factor (PDGF) by regulating cell polarization. FAK depletion attenuated myosin activation without affecting other signaling pathways. The presentation explored how FAK interacts with Dia2 and cortactin to control SMC migration and proposed future studies on these interactions and their signaling regulation.
Large hexanucleotide repeats in the C9ORF72 gene are associated with Amyotrophic Lateral Sclerosis-Frontotemporal Dementia (ALS-FTD). Zebrafish models with C9ORF72 knockout show motor neuron defects but have limitations in representing human disease. This study aims to generate human ALS-FTD induced pluripotent stem cells (iPSCs) and use them to model the disease. The TALEN system will be used to alter hexanucleotide repeat numbers in iPSCs to examine the relationship between repeat expansions and RNA expression changes in human neurons in order to better understand ALS-FTD pathogenesis.
Increasing the efficiency of homology-directed repair for crispr-cas9-induced...Hamza Khan
This document discusses using CRISPR-Cas9 gene editing to precisely modify genes in mammalian cells. It finds that suppressing non-homologous end joining (NHEJ) DNA repair increases the efficiency of homology-directed repair (HDR) for precise gene editing. Knocking down proteins involved in NHEJ, like KU70, KU80, and DNA ligase IV, increases the fraction of cells repaired by HDR up to 5-fold. Inhibiting ligase IV with SCR7 or expressing adenovirus proteins also increases HDR up to 7-fold while decreasing NHEJ repairs. This enhanced HDR could enable more efficient gene correction and targeted mutagenesis in cells
This document provides an overview of non-coding RNA (ncRNA), including long ncRNAs and small ncRNAs. It discusses different types of small ncRNAs such as microRNAs (miRNAs), piRNAs, and small interfering RNAs (siRNAs). The document describes the biogenesis and mechanisms of action of these ncRNAs. It provides examples of functions for long ncRNAs and the role of the siRNA pathway in preventing transgenerational retrotransposition in plants under stress. In summary, the document categorizes and explains the major types and roles of coding and non-coding RNAs.
The document discusses the importance of microRNAs and their role in various biological processes and diseases. It notes that microRNAs are involved in processes like cell cycle control, apoptosis, stem cell differentiation, and development. They also serve as diagnostic biomarkers for diseases like cancer and heart disease. Extracellular microRNAs can be transported between cells via exosomes, microvesicles, apoptotic bodies, and through binding to proteins. Dysregulation of microRNAs is related to several human diseases, including inherited diseases, cancer, heart disease, and kidney disease. MicroRNAs have potential roles as diagnostic markers, in influencing disease progression, and as targets for new treatment approaches.
Cardiovascular disease remains a major cause of death worldwide. Noncoding RNAs such as microRNAs play an important regulatory role in cardiovascular disease by controlling gene expression. Recent research has identified several microRNAs that are differentially expressed in cardiovascular conditions like myocardial infarction and coronary heart disease and may serve as biomarkers. Further study of microRNAs may help develop new diagnostic and therapeutic approaches for cardiovascular disease.
Will Jackson presented his Master's defense talk on identifying interactions between the transcription factor Lhx2 and other neocortical proteins. He used a yeast two-hybrid screen to identify interacting proteins, finding that Lhx2 interacts with the RNA helicase Ddx50 and exonuclease Eri3. Further experiments showed the LIM domain of Lhx2 is necessary for binding to Eri3. These interactions may regulate transcription and mRNA stability during neocortical development. Future work will aim to clarify the functional roles and pathways of Lhx2 with its interacting partners.
The document summarizes a thesis defense presentation on the role of focal adhesion kinase (FAK) in vascular smooth muscle cell (SMC) migration. The results showed that FAK mediates SMC migration toward platelet-derived growth factor (PDGF) by regulating cell polarization. FAK depletion attenuated myosin activation without affecting other signaling pathways. The presentation explored how FAK interacts with Dia2 and cortactin to control SMC migration and proposed future studies on these interactions and their signaling regulation.
Large hexanucleotide repeats in the C9ORF72 gene are associated with Amyotrophic Lateral Sclerosis-Frontotemporal Dementia (ALS-FTD). Zebrafish models with C9ORF72 knockout show motor neuron defects but have limitations in representing human disease. This study aims to generate human ALS-FTD induced pluripotent stem cells (iPSCs) and use them to model the disease. The TALEN system will be used to alter hexanucleotide repeat numbers in iPSCs to examine the relationship between repeat expansions and RNA expression changes in human neurons in order to better understand ALS-FTD pathogenesis.
Increasing the efficiency of homology-directed repair for crispr-cas9-induced...Hamza Khan
This document discusses using CRISPR-Cas9 gene editing to precisely modify genes in mammalian cells. It finds that suppressing non-homologous end joining (NHEJ) DNA repair increases the efficiency of homology-directed repair (HDR) for precise gene editing. Knocking down proteins involved in NHEJ, like KU70, KU80, and DNA ligase IV, increases the fraction of cells repaired by HDR up to 5-fold. Inhibiting ligase IV with SCR7 or expressing adenovirus proteins also increases HDR up to 7-fold while decreasing NHEJ repairs. This enhanced HDR could enable more efficient gene correction and targeted mutagenesis in cells
This document provides an overview of non-coding RNA (ncRNA), including long ncRNAs and small ncRNAs. It discusses different types of small ncRNAs such as microRNAs (miRNAs), piRNAs, and small interfering RNAs (siRNAs). The document describes the biogenesis and mechanisms of action of these ncRNAs. It provides examples of functions for long ncRNAs and the role of the siRNA pathway in preventing transgenerational retrotransposition in plants under stress. In summary, the document categorizes and explains the major types and roles of coding and non-coding RNAs.
The document discusses the importance of microRNAs and their role in various biological processes and diseases. It notes that microRNAs are involved in processes like cell cycle control, apoptosis, stem cell differentiation, and development. They also serve as diagnostic biomarkers for diseases like cancer and heart disease. Extracellular microRNAs can be transported between cells via exosomes, microvesicles, apoptotic bodies, and through binding to proteins. Dysregulation of microRNAs is related to several human diseases, including inherited diseases, cancer, heart disease, and kidney disease. MicroRNAs have potential roles as diagnostic markers, in influencing disease progression, and as targets for new treatment approaches.
Cardiovascular disease remains a major cause of death worldwide. Noncoding RNAs such as microRNAs play an important regulatory role in cardiovascular disease by controlling gene expression. Recent research has identified several microRNAs that are differentially expressed in cardiovascular conditions like myocardial infarction and coronary heart disease and may serve as biomarkers. Further study of microRNAs may help develop new diagnostic and therapeutic approaches for cardiovascular disease.
Effects of modifying backbone flexibility in α3 subunits of nicotinic acetylc...Victoria Dorich
A summary of my undergraduate research conducted in the Wells Lab my junior year presented at the 2013 Biochemistry and Genetics Society Poster Session.
Receptor targeted polyplexes for pdna and sirna delivery emadiElaheh Emadi- Andani
This document discusses receptor-targeted polyplexes for delivering plasmid DNA (pDNA) and small interfering RNA (siRNA). It describes the challenges of nucleic acid delivery, including degradation and immune responses. Nonviral vectors like polyplexes are formed through electrostatic interactions between nucleic acids and polymers. Cellular delivery involves binding ligands for targeting, endocytosis, endosomal escape, and intracellular trafficking. Strategies to improve delivery include PEGylation, proton sponge polymers, lysosomotropic agents, and fusogenic peptides. The document compares delivery methods and considers optimization of polyplex properties, targeting, and intracellular release.
This document summarizes Richard Rodriguez's final lab report on analyzing the L34P mutation in the gap junction protein Cx31 using site-directed mutagenesis. Key findings include:
1) The L34P mutation is associated with Erythrokeratoderma vairabilis (EKV) skin disorder and causes the disease through a recessive inheritance pattern by preventing formation of normal gap junctions between keratinocytes.
2) Site-directed mutagenesis was used to introduce the L34P mutation into a Cx31 plasmid, which was then transformed into E. coli cells. Over 200 colonies grew on each transformation plate.
3) The mutated Cx31 plasmid will be used
SiRNA Delivery for Cancer Therapy: Challenges and Future Perspective by Suvadeep Sen in Advancements in Bioequivalence & Bioavailability
https://crimsonpublishers.com/abb/fulltext/ABB.000518.php
MicroRNAs play important roles in regulating key cellular processes like the cell cycle, apoptosis, and differentiation by targeting oncogenes and tumor suppressor genes. Certain microRNAs are commonly misexpressed in cancers and can serve as diagnostic or prognostic biomarkers. Some microRNAs act as oncogenes by being overexpressed in cancers, making them potential therapeutic targets, while other underexpressed microRNAs like let-7 have tumor suppressive properties and may be developed as cancer therapies. MicroRNAs therefore represent promising candidates for improving current approaches to cancer treatment and diagnosis.
This paper presents a new computational method for efficiently identifying and studying palindromic DNA sequences in genomes. Palindromes are sequences that are the same on both DNA strands and can form cruciform structures that are associated with both beneficial and harmful cellular functions. The method uses a sliding window approach to extract all subsequences of a given length k and their reverse complements, then iteratively doubles k to find progressively longer palindromes without heuristics or false positives. The identified palindromes are stored in a searchable database to enable further exploration and multi-species analyses of patterns in palindromic DNA.
This document summarizes a study investigating how the N- and C-terminal regions of rotavirus nonstructural protein 4 (NSP4) influence its biochemical and biological properties. NSP4 is a multifunctional protein that plays a key role in rotavirus infection and virulence. The study found that cooperation between a putative amphipathic alpha-helix near the N-terminus and the C-terminal diarrhea-inducing region generates a unique conformational state in NSP4. This state promotes multimerization and enhances the protein's resistance to digestion and biological functions. Mutations disrupting the interaction between these terminal regions compromised NSP4's properties, providing insight into how its structure modulates vir
This document discusses oligonucleotides, which are short single- or double-stranded DNA or RNA molecules composed of 25 or fewer nucleotides. There are several major classes of oligonucleotide therapeutics, including antisense oligonucleotides, siRNA, miRNA, and aptamers. Antisense oligonucleotides are designed to inhibit protein expression by binding to mRNA. Chemical modifications are often made to oligonucleotides to increase stability and efficacy. Antisense oligonucleotides show promise for treating various diseases and are currently being researched for conditions such as cancer, diabetes, and neurological disorders.
microRNA for Clinical Research and Tumor AnalysisBioGenex
The discovery of microRNAs [miRNAs] has been one of the defining developments in cancer biology over the past decade. miRNAs are short, single stranded 20-22 nucleotide long, non-coding RNAs that regulate gene expression and have fundamental roles in Cancer growth and metastasis. miRNAs exert their function via base pairing with complementary mRNA molecules, resulting in gene silencing via transcriptional repression or target degradation. BioGenex solved the inherent difficulties in visualizing miRNAs in spatial context by using a propriety technology to synthesize modified, high-affinity oligonucleotides, labelling miRNA probes with multiple reporter molecules and developing a fully-integrated miRNA-ISH workflow solution allowing high throughput analysis of miRNA in the spatial context.
This document summarizes research into the RNA binding properties of DGCR8, a key protein involved in microRNA biogenesis. The main findings are:
1) DGCR8 binds various RNA substrates, including single-stranded, double-stranded, and random hairpin transcripts, with similar affinity, indicating it does not specifically recognize RNA targets.
2) NMR studies of the interaction between DGCR8 and pri-mir-16 showed intermediate exchange regimes over a wide range of stoichiometries, suggesting dynamic complex formation.
3) While DGCR8 does not discriminate between RNAs on its own, functional processing assays require both DGCR8 and Drosha, and
RrpC is an RNA-dependent RNA polymerase (RdRP) in Dictyostelium discoideum that silences the centromeric retrotransposon DIRS-1 post-transcriptionally. In deletion strains of RrpC, full-length and shorter DIRS-1 messenger RNAs accumulate, as do shorter versions of a hitherto unknown long non-coding RNA in the DIRS-1 antisense orientation. Concurrently, most small (21 mer) DIRS-1 RNAs disappear in RrpC deletion strains. RrpC is required for post-transcriptional DIRS-1 silencing and spreading of RNA silencing signals in both the 5' and 3' directions.
We previously reported a CRISPR-mediated knock-in strategy into introns of Drosophila genes, generating an attP-FRT-SA T2A-GAL4-polyA-3XP3-EGFP-FRT-attP transgenic library for multiple uses (Lee et al., 2018a). The method relied on double stranded DNA (dsDNA) homology donors with ~1 kb homology arms. Here, we describe three new simpler ways to edit genes in flies. We create single stranded DNA (ssDNA) donors using PCR and add 100 nt of homology on each side of an integration cassette, followed by enzymatic removal of one strand. Using this method, we generated GFP-tagged proteins that mark organelles in S2 cells. We then describe two dsDNA methods using cheap synthesized donors flanked by 100 nt homology arms and gRNA target sites cloned into a plasmid. Upon injection, donor DNA (1 to 5 kb) is released from the plasmid by Cas9. The cassette integrates efficiently and precisely in vivo. The approach is fast, cheap, and scalable.
Mir193b–365 is essential for brown fat differentiation by regulating genes involved in adipogenesis. The study identified Mir193b-365 as a microRNA complex necessary for brown adipose tissue differentiation. Blocking Mir193b expression inhibited brown fat marker genes, pointing to its critical role in brown fat development. Mir193b-365 associates closely with mRNAs like Prdm16 and Pparα that help upregulate it during differentiation, inducing adipogenic factors while suppressing myogenic factors.
The CRISPR/Cas9 system has emerged as one of the leading tools for modifying the genomes of organisms ranging from E. coli to humans. In this presentation, we discuss various methods for generating the crRNA and tracrRNA components that are required for guiding the Cas9 endonuclease to genomic targets. You will also learn how to optimize a new 2-part CRISPR RNA system from IDT that offers multiple benefits over other technologies.
1) The document discusses the packaging of retroviral RNA genomes, which involves the dimerization of two copies of the viral genome in the 5' untranslated region.
2) Retroviral genome packaging and dimerization are mediated by RNA packaging elements called psi sites, which typically overlap with the dimerization site.
3) The study investigated how mutations affecting the structure of stem loop C (SL-C) in the murine leukemia virus packaging element impacted genome dimerization and nucleocapsid protein binding. Mutation of SL-C to favor a "kissing interaction" inhibited dimerization but enhanced nucleocapsid binding, while a mutation preventing this interaction promoted dimerization but weakened nucleocapsid
DNA and RNA Structure
Central Dogma of Life
Protein Engineering (Brief)
Introduction to microRNA (miRNA)
History of miRNA
Biogenesis of miRNA
Conservation of miRNA
Impact of miRNA
miRNA Therapy
Conclusion
CRISPR-Cas9 is a powerful tool for genome engineering. The document provides guidance on using CRISPR-Cas9 to modify genomes. It describes: 1) Designing single guide RNAs (sgRNAs) to target specific gene loci using online tools; 2) Constructing plasmids expressing Cas9 and sgRNAs; 3) Validating plasmid function using assays like Surveyor nuclease; and 4) Transfecting cells, isolating clones, and further validating genome edits through sequencing. The goal is to use this method to precisely modify genomes for research applications.
This document discusses RNA-RNA cross talk through competing endogenous RNAs (ceRNAs). It explains that protein-coding and non-coding RNA transcripts can act as endogenous miRNA sponges or ceRNAs by competing for shared miRNAs. Many types of RNAs can have ceRNA activity, including pseudogenes, lncRNAs, circRNAs, and mRNAs. Effective ceRNA interaction depends on factors like miRNA and ceRNA expression levels, number of shared miRNA binding sites, and binding affinity. RNA editing and changes in ceRNA regulation can contribute to human disease.
This study examines the degradation pathway of the thiazide-sensitive NaCl cotransporter (NCC) in yeast and mammalian cells. The authors show that NCC is a substrate of endoplasmic reticulum-associated degradation (ERAD) in yeast. Using yeast strains with mutations in ERAD components, they identify the E3 ubiquitin ligase Hrd1 and the cytoplasmic Hsp70 chaperone Ssa1/Hsp70 as important for NCC ubiquitination and degradation. Expression of NCC in mammalian kidney cells reveals similar polyubiquitination and proteasome-dependent degradation. Cytoplasmic Hsp70 preferentially associates with immature glycosylated NCC, indicating its role
The document summarizes research on ankyrin proteins and their role in localizing membrane proteins in specialized membrane domains. Ankyrins function as adaptors that bind to membrane proteins through unstructured motifs, targeting them to axon initial segments, nodes of Ranvier, photoreceptor outer segments, cardiomyocyte intercalated discs and costameres. Knockdown of ankyrins leads to loss of localization of binding partners and disruption of these membrane domains.
Effects of modifying backbone flexibility in α3 subunits of nicotinic acetylc...Victoria Dorich
A summary of my undergraduate research conducted in the Wells Lab my junior year presented at the 2013 Biochemistry and Genetics Society Poster Session.
Receptor targeted polyplexes for pdna and sirna delivery emadiElaheh Emadi- Andani
This document discusses receptor-targeted polyplexes for delivering plasmid DNA (pDNA) and small interfering RNA (siRNA). It describes the challenges of nucleic acid delivery, including degradation and immune responses. Nonviral vectors like polyplexes are formed through electrostatic interactions between nucleic acids and polymers. Cellular delivery involves binding ligands for targeting, endocytosis, endosomal escape, and intracellular trafficking. Strategies to improve delivery include PEGylation, proton sponge polymers, lysosomotropic agents, and fusogenic peptides. The document compares delivery methods and considers optimization of polyplex properties, targeting, and intracellular release.
This document summarizes Richard Rodriguez's final lab report on analyzing the L34P mutation in the gap junction protein Cx31 using site-directed mutagenesis. Key findings include:
1) The L34P mutation is associated with Erythrokeratoderma vairabilis (EKV) skin disorder and causes the disease through a recessive inheritance pattern by preventing formation of normal gap junctions between keratinocytes.
2) Site-directed mutagenesis was used to introduce the L34P mutation into a Cx31 plasmid, which was then transformed into E. coli cells. Over 200 colonies grew on each transformation plate.
3) The mutated Cx31 plasmid will be used
SiRNA Delivery for Cancer Therapy: Challenges and Future Perspective by Suvadeep Sen in Advancements in Bioequivalence & Bioavailability
https://crimsonpublishers.com/abb/fulltext/ABB.000518.php
MicroRNAs play important roles in regulating key cellular processes like the cell cycle, apoptosis, and differentiation by targeting oncogenes and tumor suppressor genes. Certain microRNAs are commonly misexpressed in cancers and can serve as diagnostic or prognostic biomarkers. Some microRNAs act as oncogenes by being overexpressed in cancers, making them potential therapeutic targets, while other underexpressed microRNAs like let-7 have tumor suppressive properties and may be developed as cancer therapies. MicroRNAs therefore represent promising candidates for improving current approaches to cancer treatment and diagnosis.
This paper presents a new computational method for efficiently identifying and studying palindromic DNA sequences in genomes. Palindromes are sequences that are the same on both DNA strands and can form cruciform structures that are associated with both beneficial and harmful cellular functions. The method uses a sliding window approach to extract all subsequences of a given length k and their reverse complements, then iteratively doubles k to find progressively longer palindromes without heuristics or false positives. The identified palindromes are stored in a searchable database to enable further exploration and multi-species analyses of patterns in palindromic DNA.
This document summarizes a study investigating how the N- and C-terminal regions of rotavirus nonstructural protein 4 (NSP4) influence its biochemical and biological properties. NSP4 is a multifunctional protein that plays a key role in rotavirus infection and virulence. The study found that cooperation between a putative amphipathic alpha-helix near the N-terminus and the C-terminal diarrhea-inducing region generates a unique conformational state in NSP4. This state promotes multimerization and enhances the protein's resistance to digestion and biological functions. Mutations disrupting the interaction between these terminal regions compromised NSP4's properties, providing insight into how its structure modulates vir
This document discusses oligonucleotides, which are short single- or double-stranded DNA or RNA molecules composed of 25 or fewer nucleotides. There are several major classes of oligonucleotide therapeutics, including antisense oligonucleotides, siRNA, miRNA, and aptamers. Antisense oligonucleotides are designed to inhibit protein expression by binding to mRNA. Chemical modifications are often made to oligonucleotides to increase stability and efficacy. Antisense oligonucleotides show promise for treating various diseases and are currently being researched for conditions such as cancer, diabetes, and neurological disorders.
microRNA for Clinical Research and Tumor AnalysisBioGenex
The discovery of microRNAs [miRNAs] has been one of the defining developments in cancer biology over the past decade. miRNAs are short, single stranded 20-22 nucleotide long, non-coding RNAs that regulate gene expression and have fundamental roles in Cancer growth and metastasis. miRNAs exert their function via base pairing with complementary mRNA molecules, resulting in gene silencing via transcriptional repression or target degradation. BioGenex solved the inherent difficulties in visualizing miRNAs in spatial context by using a propriety technology to synthesize modified, high-affinity oligonucleotides, labelling miRNA probes with multiple reporter molecules and developing a fully-integrated miRNA-ISH workflow solution allowing high throughput analysis of miRNA in the spatial context.
This document summarizes research into the RNA binding properties of DGCR8, a key protein involved in microRNA biogenesis. The main findings are:
1) DGCR8 binds various RNA substrates, including single-stranded, double-stranded, and random hairpin transcripts, with similar affinity, indicating it does not specifically recognize RNA targets.
2) NMR studies of the interaction between DGCR8 and pri-mir-16 showed intermediate exchange regimes over a wide range of stoichiometries, suggesting dynamic complex formation.
3) While DGCR8 does not discriminate between RNAs on its own, functional processing assays require both DGCR8 and Drosha, and
RrpC is an RNA-dependent RNA polymerase (RdRP) in Dictyostelium discoideum that silences the centromeric retrotransposon DIRS-1 post-transcriptionally. In deletion strains of RrpC, full-length and shorter DIRS-1 messenger RNAs accumulate, as do shorter versions of a hitherto unknown long non-coding RNA in the DIRS-1 antisense orientation. Concurrently, most small (21 mer) DIRS-1 RNAs disappear in RrpC deletion strains. RrpC is required for post-transcriptional DIRS-1 silencing and spreading of RNA silencing signals in both the 5' and 3' directions.
We previously reported a CRISPR-mediated knock-in strategy into introns of Drosophila genes, generating an attP-FRT-SA T2A-GAL4-polyA-3XP3-EGFP-FRT-attP transgenic library for multiple uses (Lee et al., 2018a). The method relied on double stranded DNA (dsDNA) homology donors with ~1 kb homology arms. Here, we describe three new simpler ways to edit genes in flies. We create single stranded DNA (ssDNA) donors using PCR and add 100 nt of homology on each side of an integration cassette, followed by enzymatic removal of one strand. Using this method, we generated GFP-tagged proteins that mark organelles in S2 cells. We then describe two dsDNA methods using cheap synthesized donors flanked by 100 nt homology arms and gRNA target sites cloned into a plasmid. Upon injection, donor DNA (1 to 5 kb) is released from the plasmid by Cas9. The cassette integrates efficiently and precisely in vivo. The approach is fast, cheap, and scalable.
Mir193b–365 is essential for brown fat differentiation by regulating genes involved in adipogenesis. The study identified Mir193b-365 as a microRNA complex necessary for brown adipose tissue differentiation. Blocking Mir193b expression inhibited brown fat marker genes, pointing to its critical role in brown fat development. Mir193b-365 associates closely with mRNAs like Prdm16 and Pparα that help upregulate it during differentiation, inducing adipogenic factors while suppressing myogenic factors.
The CRISPR/Cas9 system has emerged as one of the leading tools for modifying the genomes of organisms ranging from E. coli to humans. In this presentation, we discuss various methods for generating the crRNA and tracrRNA components that are required for guiding the Cas9 endonuclease to genomic targets. You will also learn how to optimize a new 2-part CRISPR RNA system from IDT that offers multiple benefits over other technologies.
1) The document discusses the packaging of retroviral RNA genomes, which involves the dimerization of two copies of the viral genome in the 5' untranslated region.
2) Retroviral genome packaging and dimerization are mediated by RNA packaging elements called psi sites, which typically overlap with the dimerization site.
3) The study investigated how mutations affecting the structure of stem loop C (SL-C) in the murine leukemia virus packaging element impacted genome dimerization and nucleocapsid protein binding. Mutation of SL-C to favor a "kissing interaction" inhibited dimerization but enhanced nucleocapsid binding, while a mutation preventing this interaction promoted dimerization but weakened nucleocapsid
DNA and RNA Structure
Central Dogma of Life
Protein Engineering (Brief)
Introduction to microRNA (miRNA)
History of miRNA
Biogenesis of miRNA
Conservation of miRNA
Impact of miRNA
miRNA Therapy
Conclusion
CRISPR-Cas9 is a powerful tool for genome engineering. The document provides guidance on using CRISPR-Cas9 to modify genomes. It describes: 1) Designing single guide RNAs (sgRNAs) to target specific gene loci using online tools; 2) Constructing plasmids expressing Cas9 and sgRNAs; 3) Validating plasmid function using assays like Surveyor nuclease; and 4) Transfecting cells, isolating clones, and further validating genome edits through sequencing. The goal is to use this method to precisely modify genomes for research applications.
This document discusses RNA-RNA cross talk through competing endogenous RNAs (ceRNAs). It explains that protein-coding and non-coding RNA transcripts can act as endogenous miRNA sponges or ceRNAs by competing for shared miRNAs. Many types of RNAs can have ceRNA activity, including pseudogenes, lncRNAs, circRNAs, and mRNAs. Effective ceRNA interaction depends on factors like miRNA and ceRNA expression levels, number of shared miRNA binding sites, and binding affinity. RNA editing and changes in ceRNA regulation can contribute to human disease.
This study examines the degradation pathway of the thiazide-sensitive NaCl cotransporter (NCC) in yeast and mammalian cells. The authors show that NCC is a substrate of endoplasmic reticulum-associated degradation (ERAD) in yeast. Using yeast strains with mutations in ERAD components, they identify the E3 ubiquitin ligase Hrd1 and the cytoplasmic Hsp70 chaperone Ssa1/Hsp70 as important for NCC ubiquitination and degradation. Expression of NCC in mammalian kidney cells reveals similar polyubiquitination and proteasome-dependent degradation. Cytoplasmic Hsp70 preferentially associates with immature glycosylated NCC, indicating its role
The document summarizes research on ankyrin proteins and their role in localizing membrane proteins in specialized membrane domains. Ankyrins function as adaptors that bind to membrane proteins through unstructured motifs, targeting them to axon initial segments, nodes of Ranvier, photoreceptor outer segments, cardiomyocyte intercalated discs and costameres. Knockdown of ankyrins leads to loss of localization of binding partners and disruption of these membrane domains.
1. The document discusses lateral root development in Arabidopsis thaliana, including the initiation and stages of lateral root primordia formation. Key processes like cell division and emergence are described.
2. Lateral root development is regulated by auxin and other signals in a transcriptional cascade. Auxin binds to repressor proteins and activates transcription factors that regulate genes involved in cell wall remodeling and lateral root primordia development.
3. The document also covers root gravitropism, describing the three phases of gravity perception in columella cells, signal transduction through auxin transport, and the gravitropic response through differential cell elongation. Many genes involved in these processes remain unidentified.
1) The study investigates how localized protein translation in axons regulates presynaptic development at synapses.
2) The researchers developed a method to selectively repress cap-dependent translation in axons using a targeted translational repressor.
3) They found that repressing axonal translation enlarged synaptic vesicle recycling pools, and this effect was partly due to decreased levels of p35, a protein involved in regulating vesicle recycling pools. Local translation of p35 mRNA in axons normally helps regulate vesicle recycling.
This study aimed to clone homologous genes of SND1, a key regulator of secondary cell wall biosynthesis, from Populus trichocarpa. The researcher amplified four SND1 homologs from P. trichocarpa cDNA using PCR. The amplified genes were cloned into E. coli and sequenced. Two colonies were found to contain the correct SND1 sequence insert, while others were false positives. Further work will express and quantify the proteins and determine their effects on other genes and phenotypes.
This project aims to develop pharmacological agents that block T-type calcium channels (Cav3.2) for therapeutic applications. Rabbits were immunized with a peptide from the extracellular loop between transmembrane segments 3 and 4 of Cav3.2. Antibodies from rabbit serum showed immunoreactivity against the peptide. These antibodies will be tested for their ability to bind and block Cav3.2 channels expressed in HEK293 cells using techniques like western blotting and patch clamping. Successful blocking antibodies may aid in understanding Cav3.2 function and developing drugs targeting voltage-gated calcium channels.
CRISPR is a powerful new tool for genome editing that allows targeted modifications to genes. It utilizes the Cas9 enzyme to cut DNA at a specific site guided by a short RNA molecule. This summary will discuss the history and mechanisms of CRISPR/Cas9 and its applications in biotechnology and agriculture. CRISPR represents a major breakthrough that will revolutionize genetic engineering by enabling precise edits to genomes. However, further refinement is needed to address issues such as off-target effects. Overall, CRISPR technology holds tremendous promise for developing improved crop traits.
Light Regulates Plant Alternative Splicing through the Control of Transcripti...ShreyaMandal4
This document discusses how light regulates alternative splicing in plants through controlling transcription elongation. It presents a study that investigated the effects of light-dark conditions and histone deacetylase inhibitors on alternative splicing in Arabidopsis seedlings. The study found that light increases RNA polymerase II elongation, which regulates alternative splicing. Light-dark conditions affected alternative splicing but not total mRNA levels. The results suggest that kinetic coupling between transcription and alternative splicing is an important mechanism for plants to respond to environmental cues like light.
This document discusses treatment response assessment for lymphoma. The original International Working Group (IWG) criteria from 1999 defined response categories based on CT imaging of lymph node size, but had limitations. An updated 2007 version by IWG integrated 18FDG-PET, immunohistochemistry, and flow cytometry to add sensitivity to staging and evaluating treatment response. The 2014 Lugano classification highlights include recommending 18FDG-PET/CT for response assessment in FDG-avid lymphomas using a 5-point scale and preferring CT for non-FDG-avid types. Overall the document outlines improvements made to standards for assessing treatment response in lymphoma patients.
This document summarizes the findings of a study investigating the role of chromosomal contacts in regulating the coordinated expression of genes within multigene complexes. The study finds that disruption of chromosomal contacts via TALEN-induced DNA breaks leads to changes in the transcriptional hierarchy of genes within a complex. Specifically, disruption of the SAMD4A gene abrogates expression of TNFAIP2 and SLC6A5, but disruption of TNFAIP2 only impacts SLC6A5 expression. Restoration of the SAMD4A gene contact rescues coordinated expression across the complex. However, the study does not rule out the possibility that direct transcriptional tethering, rather than contact alone, regulates expression. Overall,
P68 RNA helicase unwinds the human let-7 microRNA precursor duplex and is req...David W. Salzman
P68 RNA helicase was identified as being required for unwinding the human let-7 microRNA precursor duplex. Recombinant P68 RNA helicase was shown to unwind the let-7 duplex in vitro. Knockdown of P68 inhibited let-7 microRNA function, indicating P68 is essential for loading let-7 into the silencing complex. This study identifies P68 RNA helicase as playing a key role in the human let-7 microRNA pathway.
Global run-on sequencing (GRO-Seq) is a method to map the binding sites of transcriptionally active RNA polymerase II. It involves allowing RNA polymerase II to actively transcribe in the presence of labeled nucleotides, followed by purification and sequencing of the newly synthesized RNA. This provides sequences of RNAs that are currently being transcribed, without prior knowledge of transcription sites. While it directly determines relative transcriptional activity, GRO-Seq is limited to cell cultures and may introduce artifacts during nuclear preparation or transcription run-on.
1. LncRNAs interact with chromatin and regulate gene expression through several mechanisms including chromatin regulation, protein-lncRNA localization and function on chromatin, and direct interactions between lncRNA and DNA.
2. LncRNAs can interact with chromatin through mechanisms like chromatin decompaction, altering the chromatin environment, and binding DNA directly or through protein interactions. This regulates gene expression in cis or trans.
3. Direct lncRNA-DNA interactions like triplex structures and R-loops also influence chromatin accessibility and gene expression by recruiting chromatin modifiers or transcription factors.
Phosphoproteomics of collagen receptor networks reveals SHP-2Maciej Luczynski
This document describes a study that used phosphoproteomics to analyze signaling networks downstream of collagen receptors. The researchers identified 424 phosphorylated proteins over seven time points after stimulating cells with collagen. Multiple clustering analysis revealed that phosphorylation sites on proteins like SHP-2, NCK1, LYN, and PIK3C2A strongly clustered with DDR2 phosphorylation dynamics, suggesting these proteins are candidate downstream effectors of DDR2 signaling. Biochemical validation showed SHP-2 tyrosine phosphorylation depends on DDR2 kinase activity. Targeted proteomics of lung cancer DDR2 mutants showed SHP-2 is phosphorylated by some mutants. This indicates SHP-2 is a key signaling node downstream of DDR
This document summarizes an experiment aiming to knockout the CTNNB1 gene in mouse embryonic stem cells using CRISPR-Cas9 genome editing. It describes designing a guide RNA targeting exon 10 of CTNNB1, cloning it into a vector, transforming E. coli, and testing primers for detecting knockout via PCR and qPCR. Prior studies showed CTNNB1 knockout embryos had defects in ectoderm and mesoderm development. The authors hypothesized knocking out CTNNB1 in stem cells would produce inviable embryos, similar to prior findings.
The nuclear envelope contains an inner and outer membrane, separated by the perinuclear space. It regulates transport between the nucleus and cytoplasm. The nuclear lamina lies underneath the inner nuclear membrane and provides structural support. Nuclear pore complexes control molecular traffic through the envelope. Small molecules can passively diffuse, while larger cargoes require transport receptors and Ran GTPase. Importins ferry proteins into the nucleus via nuclear localization signals, while exportins use nuclear export signals and RanGTP to transport proteins and RNAs out of the nucleus. This ensures proper distribution and functions of biomolecules in the cell.
1. The document discusses systems biology approaches to modeling the endothelial cell response to fluid shear stress. It describes experimental techniques to apply controlled fluid shear stress and measure downstream cellular responses.
2. Mathematical models are formulated to represent molecular interactions and pathways involved in the shear stress response. Model predictions are compared to experimental data to validate and refine the models.
3. The models can provide insights into critical components, feedback loops, and how external perturbations may influence the cellular response to shear stress. Further experimental validation of model predictions is needed.
MALAT1 is a long non-coding RNA that is overexpressed in many cancers and associated with poor prognosis. It plays roles in alternative splicing, transcriptional regulation, acting as a ceRNA, and conferring drug resistance. Specifically, it localizes to nuclear speckles and regulates alternative splicing by interacting with splicing factors like SR proteins. It also interacts with PRC2 and recruits it to target genes to repress transcription. Additionally, MALAT1 can act as a ceRNA by sponging various miRNAs to regulate genes involved in processes like migration, invasion, and stemness.
This document summarizes research on mitochondrial DNA (mtDNA) and its mutation rate. It discusses how mtDNA is inherited maternally and generally does not recombine. Two methods for estimating mtDNA mutation rates are pedigree analysis and phylogenetic analysis, which produce rates that differ by around 3 times. Both rates provide accurate estimates of divergence times depending on how closely or distantly sequences are related. The document also explores evidence of mtDNA recombination in one individual who inherited mitochondria from both parents.
1) Dendritically localized transcripts in hippocampal neurons form distinct ribonucleoprotein (RNP) particles that display fast, directional motility along dendrites.
2) Microinjection experiments show that fluorescently labeled RNAs localize as expected and maintain their localization patterns even when multiple tagged RNAs are present, suggesting specificity of dendritic versus non-dendritic particles.
3) Tracking of particle movement reveals a range of speeds from 0.2 to 1.2 μm/sec, suggesting motor-dependent transport of RNP particles along microtubules in dendrites.
3. Overview Introduction Neuronal polarity and polarized trafficking pathways Parts 1 & 2: Polarized protein trafficking: Are SNARE proteins polarized in neurons, and are they involved in polarized trafficking of neuronal membrane proteins? Ion channel localization: Are trafficking proteins associated with ion channels, and do they play a role in their localization and clustering?
16. MT Golgi Nucleus Plasma membrane syntaxins MDCK polarized epithelial cells: Syntaxin 3: apical membrane. Syntaxin 3 F31A mutant: non-polarized. Syntaxin 4: basolateral membrane. (Low et al., 1998; Low et al., 2006; Sharma et al.,2006) Apical Different syntaxin distribution suggests that these syntaxins may participate in specifying fusion of vesicles to specific compartments. Question: Are these syntaxins involved in specifying the targeting of selective proteins to specific neuronal domains?
17. Syntaxins 3 and 4 are expressed in hippocampal neurons NRK = Normal rat kidney cells Syntaxin 4 Syntaxin 3 Question: Are the plasma membrane syntaxins Stx3 and Stx4 polarized in neurons?
18. Syntaxin 3 Localizes to Axon and Neurite Tips Total Syntaxin 3 Total Syntaxin 3 GFP
21. N-terminal targeting motif is required for polarized syntaxin 3 trafficking Stx3Δ38 GFP Stx3AAA GFP axon dendrites axon axon
22. Is syntaxin 3 responsible for polarized trafficking of vesicles carrying cargo to the axons?
23. NgCAM (L1)—an axonal targeted cargo NgCAM (L1) is a neural cell adhesion molecule, a member of the immunoglobulin superfamily, that is expressed in axons and growth cones. Intracellular NgCAM is expressed in a non-polarized manner in hippocampal neurons. NgCAM surface expression is highly polarized to the axon. This suggests that selective fusion is the mechanism involved in axonal polarity of NgCAM.
26. Syntaxin 3 does not affect trafficking of a dendritic cargo transferrin receptor
27.
28. Dendritic proteins have low Polarity Index RatiosN =25-30 cells Polarity Index: Ratio of surface NgCAM (axon) surface NgCAM (dendrite) Conclusion: Syntaxin 3 is required for mediating targeting of axonal cargo protein and not dendritic cargo protein
54. GST-pulldowns from rat brains Kir2.3 C-terminal domain interacts with MAGUK PSD-95 and Homer proteins Dmitri Leonoudakis, unpublished Question: Does the synaptic localization of Kir2.3 channels depend on the PDZ interaction or Homer interaction?
55. Kir2.3 clustering in spines depends on PDZ interaction Colocalization with PSD-95 n = 14-15 cells
57. si-PSD-95 Eliminates PSD-95 Clustering in Spines PSD-95 (endogenous) PSD-95 (endogenous) GFP GFP Uninfected Infected with Adeno-si-PSD-95 GFP
58. PSD-95 is Required for Channel Clustering in Dendritic Spines PSD-95 HA-Kir2.3 HA-Kir2.3 PSD-95 GFP Transfected with si-PSD-95 GFP and HA-Kir2.3 Untransfected
59.
60. Conclusions PDZ-binding motif is required for potassium channel clustering to dendritic spines. PSD-95 is required for targeting and clustering Kir2 channels to dendritic spines These studies demonstrate that Kir2 channel localization to spines depend on its association with PSD-95 via the PDZ-binding motif.
61.
62. Acknowledgements Neuroscience Research Institute Lab Members: Chris D. Banna Dmitri Leonoudakis Carolyn M. Radeke Melanie Williams Lior Dessau Lisa Conti Advisor: Dr. Carol Vandenberg Funding: Cottage Hospital Grant Muscular Dystrophy American Heart Association Collaborators: Thomas Weimbs Seng Wei Low Nikunj Sharma (UCSB)
Editor's Notes
In my talk, I will describe studies that uncover conserved mechanisms underlying membrane protein targeting. In cell biology, one the fundamental aspects of all eukaryotic cells is cell polarity. What underlies this polarity is that specific proteins and lipids are delivered to specific regions of the plasma membrane, thereby defining and maintaining their unique molecular and functional identities. This is the result of some fundamental mechanisms preserve from yeast to humans. Here I will describe work covering the neuronal trafficking behaviors of two classes of proteins– syntaxins and K+ channels. Functions:Consolidate short-term and long-term memoryEpisodic and contextual memorySpatial Orientation/NavigationDetection of novel stimuliNeurogenesisHere we show that syntaxins 3 and 4 are expressed in hippocampal neurons and that polarized expression of syntaxin 3 confers specificity to axonal protein trafficking. Syntaxin 3 localizes to the axonal plasma membrane, particularly to axonal tips, whereas syntaxin 4 localizes to the somatodendritic plasma membrane. Disruption of a conserved N-terminal targeting motif causes mislocalization of syntaxin 3, resulting in coincident mistargeting of the axonal NgCAM, but not of the somatodendritic transferrin receptor. RNAi-mediated knockdown of endogenous syntaxin 3 leads to partial mistargeting of NgCAM, demonstrating that syntaxin 3 is critical to axonal targeting. Scaffolding proteins at postsynaptic sites are crucial for organizing ion channels into signaling complexes essential for synaptic transmission. Inward rectifier potassium Kir2.3 channels are enriched at spines, when expressed in cultured hippocampal neurons, but the mechanisms underlying channel localization are unclear. We used mutational analysis to determine how protein-interacting motifs in the C-terminus of Kir2.3 influence its localization. We show that Kir2.3 channels traffic to spines and that synaptic localization requires a previously identified C-terminal PDZ (postsynaptic density-95/Discs large/zona-occludent-1)-binding motif. We also identify a polyproline motif in the C-terminus of Kir2.3 that associates with Homer-1 and Homer-2 in brain. Deleting the PDZ-binding motif abolishes binding of PSD-95 with Kir2.3 and dramatically reduces Kir2.3 synaptic localization, whereas mutating residues in the polyproline motif has little effect on channel localization. Using RNA interference, we show that PSD-95 is necessary for localizing Kir2.3 to spines.
Based on the trafficking studies using hipp neurons, a variety of mechanisms have been proposed including selective delivery (in which carrier vesicles deliver cargo exclusively to specifically to either the axonal or dendritic membrane); or selective fusion (in which cargo could be delivered to either the axonal or dendritic domains but become expressed exclusively on the surface of one by selective fusion machinery); or the expression of proteins on the correct surface domain could also be achieved by selective endocytic machinery, in which case cargo are first delivered uniformily to the surface but are selective endocytosed and subsequently delivered to the correct domain. Test the hypothesis that syntaxins are involved in specifying the targeting of membrane proteins to specific domainsuse this slide as the opportunity to describe in words what you are going to test (Selective fusion hypothesis), and what your results are going to be (Stx3 specifies selective axonal fusion)
Fusion is thought to occur when SNAREs on the vesicle, v-SNAREs, interact with those on the target membranes (t-SNAREs) and based on the zipper model they zip up into a four helical bundle that drives fusion of apposing membranes. The pairing of v-SNAREs expressed on cargo vesicles matching up with a cognate t-SNAREs on the plasma membrane was initially proposed by the SNARE hypothesis from James Rothman to contribute to specificity of membrane trafficking. This was demonstrated by in vitro reconstitution fusion assays that a successful SNARE pair lead to successful membrane fusion.
There are primarily three subclass of molecules that make up the SNARE complex. Syntaxin. Fusion. Brain. Synaptic vesicles. Isolated in late 80s and early 90s.
Not only are SNAREs important for synaptic transmission but also they are important mediators of fusion along all intracellular trafficking pathways in mammals. How is specificity achieved during membrane trafficking? In other words, Question: What enables cargo-carrying vesicles to target and deliver their cargo to specific domains (axons or dendrites) correctly?From yeast to mammals, SNAREs are required for vesicle fusion along both secretory and endocytic pathways.
Different subcellular locations of these syntaxins suggest that there is a specificity of trafficking pathways and perhaps fusion specificity of cargo carring vesicles to a specific membraneSyntaxin 4 knockout is embronic lethal but partial knockout exhibit 50% reduction in whole-body glucose uptake Apical targeting of stx3 depends on an N-terminal domain. Point mutations and chimera studies showed that this domains was indeed necessary and sufficient for apical trafficking of syntaxin 3.Since these syntaxins localize to different subcellular locations, this suggest that they might be involved in specifying fusion of cargo-laden vesicles to specific compartmentsAlthough these findings indicate that plasma membrane syntaxins are important for polarized trafficking pathways in epithelial cells, whether selective fusion of vesicles carrying cargo is involved in neuronal polarity and selective targeting of proteins is unknown. Here I wanted to test the hypothesis that syntaxins are involved in specifying the
Before we get to that question, we wanted to first ask whether syn 3 and 4 were expressed in our hipp cutlures. By RT-PCR done by Nikunj as shown here on the left and by western blotting on the right, syntaxin 3 and 4 were found endogenously expressed in our cultured neurons. We found that there were 2 isoforms A and B of syn3 expressed in neurons, whereas only the A isoform was present in normal rat kidney cell lines. Similarly in westerns, both syn 3 and 4 are again expressed from cultured neurons as well as adult hippocampus homogenates similar to syn1Next, we wanted to ask where syn3 and 4 were polarized in neurons?
One of first clues came from Nikunj studies in epithelial cells where he showed that an N-terminal motif: FMDEFF was found to be critical for its targeting to the apical membrane in epithelial cells. We thought that would be good place to start so several constructs were made including a mutant where the N-terminus containing this motif was removed and another mutant where there amino acids were individually mutated to alanine. when they mutated the F31, E34, and F36 individually to alanine.
When I expressed these syntaxin 3 mutants in neurons I found that
Now that we were able to show that Stx3 and Stx4 are polarized in neurons, and that it is possible to mislocalize Stx3, we were able to address the key question of whether selective membrane fusion contributes to polarized neuronal trafficking. The selective fusion model predicts that if the specific SNARE protein is moved to a new location in the cell, then the cargo will fuse at the new location. The overall strategy to test this was to use an axonal cargo and a dendritic cargo, and ask whether these cargoes could be re-routed by mislocalization of syntaxin 3.
One attractive candidate as an axonal cargo was NgCAM, Live cell imaging studies done in Gary banker’s lab (Burack 2000) shown NgCAM go to surface but internally was in both axon and dendrites.
When we quantified the results by measuring the intensities of surface proteins in axons over dendrites to determine the polarity index, the majority of the cells showed that syn3 mislocalization caused NgCAM to also mislocalize.
as a mixture of shRNAs gives effective knockdown
Given that SNAREs are involved in the fusion of vesicles, we wanted to test whether overexpression of syntaxins might lead to an increase in delivery of lipid molecules.
Transport K+ ions in muscle and kidney
Rare genetic disorderInherited autosomal dominant Affects heart, small lower jaw, abnormal curvature of fingers and toes
Now that we were able to show that Stx3 and Stx4 are polarized in neurons, and that it is possible to mislocalize Stx3, we were able to address the key question of whether selective membrane fusion contributes to polarized neuronal trafficking. The selective fusion model predicts that if the specific SNARE protein is moved to a new location in the cell, then the cargo will fuse at the new location. The overall strategy to test this was to use an axonal cargo and a dendritic cargo, and ask whether these cargoes could be re-routed by mislocalization of syntaxin 3.
Now in my second main project focused on understanding the mechanisms underlying the class of Kir2 potassium channel localization in neurons?Accumulating electrophysiological evidence indicates that Kir2 channels are critical regulators of synaptic integration and dendritic excitability in the forebrain and striatum (Day et al. 2005; Shen et al. reduce opening of Kir2 channels enhances dendritic excitability and synaptic integration. Modulation of these K+ channels can thus alter synaptic signaling events, and defective regulation could lead to neurological disease. Kir2.2/2.3 channel mediated conductance appears to regulate the activity of other types of channels HCN, thereby controlling dendritic excitability. Blocking channels lead to neurons to depolarize which led to deactivation of HCN channelsHold the dendritic membrane near the K+ equilibrium potenital and dampen responsiveness to glutamatergic excitatory synaptic transmission. Given the significance of these channels functional roles in neurotransmission, it is therefore important to understand how these channels are regulated in neurons. In the lab, we have focused on understanding just that.It is therefore not hard to imagine that improper channel localization could cause communication defects in neural circuitry underlying proper brain function. Correct location of channels at specific subcellular sites
Kir2.3 highly localized as puncta along dendrites.Co-labeling with post-synaptic marker, PSD-95, bright Kir2.3 puncta concentrated at spines.
Previous unpublished from Zach’s work in the Jan lab had already identified a Homer binding motif that is exclusively found in Kir2.3 and not in the other Kir2 isoforms.
A similar pattern of co-localization was found with Kir2.3-H, which lacks the polyproline motif, and PSD-95. By contrast, Kir2.3 constructs that lacked the PDZ binding motif (Kir2.3Δ3 and Kir2.3-HΔ3) were not recruited to synaptic sites in the presence of overexpressed PSD-95. Homer mutant (-H)PDZ mutant (delta 3)this high correlation coefficient is numerical evidence for the colocalization of the two proteins
Summary slides part 1—syntaxins and part 2—Kir2.3 and PSD-95
Here I showed you the importance of these scaffolding interactions of K+ channels in recruiting these chanenls to spines and making it possible to bring them in close proximity with other signaling molecules to regulate synaptic transmission.