scholarly journals Structure–function relation of the developing calyx of Held synapse in vivo

2020 ◽  
Vol 598 (20) ◽  
pp. 4603-4619 ◽  
Author(s):  
Martijn C. Sierksma ◽  
Johan A. Slotman ◽  
Adriaan B. Houtsmuller ◽  
J. Gerard G. Borst
Keyword(s):  
Structure Function ◽  
Calyx Of Held ◽  
Function Relation

scholarly journals Structure-function relation of the developing calyx of Held synapse in vivo

2020 ◽  
Author(s):  
Martijn C. Sierksma ◽  
Johan A. Slotman ◽  
Adriaan B. Houtsmuller ◽  
J. Gerard G. Borst
Keyword(s):  
Structure Function ◽  
Glutamate Transporters ◽  
Synaptic Strength ◽  
Target Cells ◽  
List Type ◽  
Calyx Of Held ◽  
Synaptic Terminals ◽  
Medial Nucleus ◽  
Vesicular Glutamate Transporters

AbstractIn adult rodents, a principal neuron in the medial nucleus of the trapezoid (MNTB) is generally contacted by a single, giant axosomatic terminal called the calyx of Held. How this one-on-one relation is established is still unknown, but anatomical evidence suggests that during development principal neurons are innervated by multiple calyces, which may indicate calyceal competition. However, in vivo electrophysiological recordings from principal neurons indicated that only a single strong synaptic connection forms per cell. To test whether a mismatch exists between synaptic strength and terminal size, we compared the strength of synaptic inputs with the morphology of the synaptic terminals. In vivo whole-cell recordings of the MNTB neurons from newborn Wistar rats of either sex were made while stimulating their afferent axons, allowing us to identify multiple inputs. The strength of the strongest input increased to calyceal levels in a few days across cells, while the strength of the second strongest input was stable. The recorded cells were subsequently immunolabeled for vesicular glutamate transporters (VGluT) to reveal axosomatic terminals with structured-illumination microscopy. Synaptic strength of the strongest input was correlated with the contact area of the largest VGluT cluster at the soma (r = 0.8), and no indication of a mismatch between structure and strength was observed. Together, our data agree with a developmental scheme in which one input strengthens and becomes the calyx of Held, but not with multi-calyceal competition.Key points summaryDuring development the giant, auditory calyx of Held forms a one-to-one connection with a principal neuron of the medial nucleus of the trapezoid body.While anatomical studies described that most of the target cells are temporarily contacted by multiple calyces, multi-calyceal innervation was only sporadically observed in in vivo recordings, suggesting a structure-function discrepancy.We correlated synaptic strength of inputs, identified in in vivo recordings, with post hoc labeling of the recorded neuron and synaptic terminals containing vesicular glutamate transporters (VGluT).During development only one input increased to the level of the calyx of Held synapse, and its strength correlated with the large VGluT cluster contacting the postsynaptic soma.As neither competing strong inputs nor multiple large VGluT clusters on a single cell were observed, our findings did not indicate a structure-function discrepancy.

Linalool Effect on HepG2 cells: Structure Function Relation

Planta Medica ◽  
2011 ◽  
Vol 77 (12) ◽  
Author(s):  
J Usta ◽  
K Racha ◽  
K Boushra ◽  
S Shatha ◽  
B Yolla ◽  
...  
Keyword(s):  
Structure Function ◽  
Hepg2 Cells ◽  
Function Relation

Mouse models to study von Willebrand factor structure-function relationships in vivo

2009 ◽  
Vol 29 (01) ◽  
pp. 17-20 ◽  
Author(s):  
I. Marx ◽  
I. Badirou ◽  
R. Pendu ◽  
O. Christophe ◽  
C. V. Denis
Keyword(s):  
Structure Function ◽  
Transient Expression ◽  
Von Willebrand Factor ◽  
Large Size ◽  
Mouse Hepatocytes ◽  
Function Relationship ◽  
Von Willebrand ◽  
Willebrand Factor

SummaryVon Willebrand factor (VWF) structure-function relationship has been studied only through in vitro approaches. The VWF-deficient mouse model has been extremely useful to examine the in vivo function of VWF but does not allow a more subtle analysis of the relative importance of its different domains. However, considering the large size of VWF and its capacity to interact with various ligands in order to support platelet adhesion and aggregation, the necessity to evaluate independently these interactions appeared increasingly crucial. A recently developed technique, known as hydrodynamic injection, which allows transient expression of a transgene by mouse hepatocytes, proved very useful in this regard. Indeed, transient expression of various VWF mutants in VWF-deficient mice contributed to improve our knowledge about the role of VWF interaction with subendothelial collagens and with platelets receptors in VWF roles in haemostasis and thrombosis. These findings can provide new leads in the development of anti-thrombotic therapies.

Hirudins: Structure-function relationships — behavior and effects in in vivo-models

1987 ◽  
Vol 45 ◽  
pp. 40
Author(s):  
H. Fritz ◽  
J. Dodt ◽  
U. Seemüller ◽  
R. Gemmerli ◽  
J. Bichler ◽  
...  
Keyword(s):  
Structure Function ◽  
In Vivo Models

scholarly journals Investigating structure function relationships in the NOTCH family through large-scale somatic DNA sequencing studies

2020 ◽  
Author(s):  
Michael W J Hall ◽  
David Shorthouse ◽  
Philip H Jones ◽  
Benjamin A Hall
Keyword(s):  
Dna Sequencing ◽  
Structure Function ◽  
Calcium Binding ◽  
Large Scale ◽  
Driver Mutations ◽  
Missense Mutations ◽  
Mutant Selection ◽  
Ligand Interaction ◽  
Binding Interface

AbstractThe recent development of highly sensitive DNA sequencing techniques has detected large numbers of missense mutations of genes, including NOTCH1 and 2, in ageing normal tissues. Driver mutations persist and propagate in the tissue through a selective advantage over both wild-type cells and alternative mutations. This process of selection can be considered as a large scale, in vivo screen for mutations that increase clone fitness. It follows that the specific missense mutations that are observed in individual genes may offer us insights into the structure-function relationships. Here we show that the positively selected missense mutations in NOTCH1 and NOTCH2 in human oesophageal epithelium cause inactivation predominantly through protein misfolding. Once these mutations are excluded, we further find statistically significant evidence for selection at the ligand binding interface and calcium binding sites. In this, we observe stronger evidence of selection at the ligand interface on EGF12 over EGF11, suggesting that in this tissue EGF12 may play a more important role in ligand interaction. Finally, we show how a mutation hotspot in the NOTCH1 transmembrane helix arises through the intersection of both a high mutation rate and residue conservation. Together these insights offer a route to understanding the mechanism of protein function through in vivo mutant selection.

scholarly journals Rapid and Programmable Protein Mutagenesis Using Plasmid Recombineering

2017 ◽  
Author(s):  
Sean A. Higgins ◽  
Sorel Ouonkap ◽  
David F. Savage
Keyword(s):  
Structure Function ◽  
Sequence Space ◽  
Protein Function ◽  
Fluorescent Protein ◽  
One Pot ◽  
Protein Mutagenesis ◽  
Protein Mutants ◽  
Protein Libraries

ABSTRACTComprehensive and programmable protein mutagenesis is critical for understanding structure-function relationships and improving protein function. However, current techniques enabling comprehensive protein mutagenesis are based on PCR and require in vitro reactions involving specialized protocols and reagents. This has complicated efforts to rapidly and reliably produce desired comprehensive protein libraries. Here we demonstrate that plasmid recombineering is a simple and robust in vivo method for the generation of protein mutants for both comprehensive library generation as well as programmable targeting of sequence space. Using the fluorescent protein iLOV as a model target, we build a complete mutagenesis library and find it to be specific and unbiased, detecting 99.8% of our intended mutations. We then develop a thermostability screen and utilize our comprehensive mutation data to rapidly construct a targeted and multiplexed library that identifies significantly improved variants, thus demonstrating rapid protein engineering in a simple one-pot protocol.

scholarly journals Oligomerization Requirements for MX2-Mediated Suppression of HIV-1 Infection

2015 ◽  
Vol 90 (1) ◽  
pp. 22-32 ◽  
Author(s):  
Matthew D. J. Dicks ◽  
Caroline Goujon ◽  
Darja Pollpeter ◽  
Gilberto Betancor ◽  
Luis Apolonia ◽  
...  
Keyword(s):  
Antiviral Activity ◽  
Structure Function ◽  
Influenza A ◽  
Acute Infection ◽  
Higher Order ◽  
Nuclear Accumulation ◽  
Antiviral Function ◽  
Ring Structures ◽  
Hiv 1

ABSTRACTHuman myxovirus resistance 2 (MX2/MXB) is an interferon-stimulated gene (ISG) and was recently identified as a late postentry suppressor of human immunodeficiency virus type 1 (HIV-1) infection, inhibiting the nuclear accumulation of viral cDNAs. Although the HIV-1 capsid (CA) protein is believed to be the viral determinant of MX2-mediated inhibition, the precise mechanism of antiviral action remains unclear. The MX family of dynamin-like GTPases also includes MX1/MXA, a well-studied inhibitor of a range of RNA and DNA viruses, including influenza A virus (FLUAV) and hepatitis B virus but not retroviruses. MX1 and MX2 are closely related and share similar domain architectures and structures. However, MX2 possesses an extended N terminus that is essential for antiviral function and confers anti-HIV-1 activity on MX1 [MX1(NMX2)]. Higher-order oligomerization is required for the antiviral activity of MX1 against FLUAV, with current models proposing that MX1 forms ring structures that constrict around viral nucleoprotein complexes. Here, we performed structure-function studies to investigate the requirements for oligomerization of both MX2 and chimeric MX1(NMX2) for the inhibition of HIV-1 infection. The oligomerization state of mutated proteins with amino acid substitutions at multiple putative oligomerization interfaces was assessed using a combination of covalent cross-linking and coimmunoprecipitation. We show that while monomeric MX2 and MX1(NMX2) mutants are not antiviral, higher-order oligomerization does not appear to be required for full antiviral activity of either protein. We propose that lower-order oligomerization of MX2 is sufficient for the effective inhibition of HIV-1.IMPORTANCEInterferon plays an important role in the control of virus replication during acute infectionin vivo. Recently, cultured cell experiments identified human MX2 as a key effector in the interferon-mediated postentry block to HIV-1 infection. MX2 is a member of a family of large dynamin-like GTPases that includes MX1/MXA, a closely related interferon-inducible inhibitor of several viruses, including FLUAV, but not HIV-1. MX GTPases form higher-order oligomeric structures, and the oligomerization of MX1 is required for inhibitory activity against many of its viral targets. Through structure-function studies, we report that monomeric mutants of MX2 do not inhibit HIV-1. However, in contrast to MX1, oligomerization beyond dimer assembly does not seem to be required for the antiviral activity of MX2, implying that fundamental differences exist between the antiviral mechanisms employed by these closely related proteins.

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