SynapsEM: Computer-Assisted Synapse Morphometry

The structural features of a synapse help determine its function. Synapses are extremely small and tightly packed with vesicles and other organelles. Visualizing synaptic structure requires imaging by electron microscopy, and the features in micrographs must be quantified, a process called morphometry. Three parameters are typically assessed from each specimen: (1) the sizes of individual vesicles and organelles; (2) the absolute number and densities of organelles; and (3) distances between organelles and key features at synapses, such as active zone membranes and dense projections. For data to be meaningful, the analysis must be repeated from hundreds to thousands of images from several biological replicates, a daunting task. Here we report a custom computer program to analyze key structural features of synapses: SynapsEM. In short, we developed ImageJ/Fiji macros to record x,y-coordinates of segmented structures. The coordinates are then exported as text files. Independent investigators can reload the images and text files to reexamine the segmentation using ImageJ. The Matlab program then calculates and reports key synaptic parameters from the coordinates. Since the values are calculated from coordinates, rather than measured from each micrograph, other parameters such as locations of docked vesicles relative to the center of an active zone can be extracted in Matlab by additional scripting. Thus, this program can accelerate the morphometry of synapses and promote a more comprehensive analysis of synaptic ultrastructure.

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SynapsEM: computer-assisted synapse morphometry

10.1101/2020.08.28.271882 ◽

2020 ◽

Author(s):

Shigeki Watanabe ◽

M Wayne Davis ◽

Grant F Kusick ◽

Janet Iwasa ◽

Erik M Jorgensen

Keyword(s):

Absolute Number ◽

Structural Features ◽

Comprehensive Analysis ◽

Computer Assisted ◽

Matlab Program ◽

Synaptic Structure ◽

Synaptic Ultrastructure ◽

Daunting Task ◽

Dense Projections ◽

Synaptic Parameters

AbstractThe structural features of a synapse, in part, determine its output. Synapses are extremely small and tightly packed with vesicles and other organelles. Visualizing synaptic structure requires imaging by electron microscopy, and the features in micrographs must be quantified using morphometry. Three parameters are typically assessed from each specimen: 1) the sizes of individual vesicles and organelles, 2) the absolute number and densities of organelles, and 3) distances between organelles and key features at synapses such as active zone membranes and dense projections. For data to be valid, the analysis must be repeated from hundreds or thousands of images from several biological replicates, a daunting task. Here we report a custom computer program to analyze these features: SynapsEM. In short, we developed macros for ImageJ/Fiji to record x,y-coordinates of segmented structures; the coordinates are then exported as text files. Independent investigators can reload the images and text files to confirm or re-evaluate the segmentation using ImageJ. The Matlab program calculates and reports key synaptic parameters from the coordinates. Since the values are calculated, rather than measured from each micrograph, other parameters can be extracted in Matlab by additional scripting. Thus, this program can accelerate morphometry of synapses and promote a more comprehensive analysis of synaptic ultrastructure.

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Mimicry between neurokinin-1 and fibronectin may explain the transport and stability of increased substance P immunoreactivity in patients with bone marrow fibrosis

Blood ◽

10.1182/blood.v97.10.3025 ◽

2001 ◽

Vol 97 (10) ◽

pp. 3025-3031 ◽

Cited By ~ 26

Author(s):

Pranela Rameshwar ◽

Deval D. Joshi ◽

Prem Yadav ◽

Jing Qian ◽

Pedro Gascon ◽

...

Keyword(s):

Bone Marrow ◽

Substance P ◽

Mononuclear Cells ◽

Cdna Probe ◽

Structural Features ◽

Cdna Libraries ◽

Future Research ◽

Computer Assisted ◽

Partial Clone ◽

Neurokinin 1

Abstract Bone marrow (BM) fibrosis may occur in myeloproliferative diseases, lymphoma, myelodysplastic syndrome, myeloma, and infectious diseases. In this study, the role of substance P (SP), a peptide with pleiotropic functions, was examined. Some of its functions—angiogenesis, fibroblast proliferation, and stimulation of BM progenitors—are amenable to inducing BM fibrosis. Indeed, a significant increase was found in SP-immunoreactivity (SP-IR) in the sera of patients with BM fibrosis (n = 44) compared with the sera of patients with hematologic disorders and no histologic evidence of fibrosis (n = 46) (140 ±12 vs 18 ±3; P < .01). Immunoprecipitation of sera SP indicated that this peptide exists in the form of a complex with other molecule(s). It was, therefore, hypothesized that SP might be complexed with NK-1, its natural receptor, or with a molecule homologous to NK-1. To address this, 3 cDNA libraries were screened that were constructed from pooled BM stroma or mononuclear cells with an NK-1 cDNA probe. A partial clone (clone 1) was retrieved that was 97% homologous to the ED-A region of fibronectin (FN). Furthermore, sequence analyses indicated that clone 1 shared significant homology with exon 5 of NK-1. Immunoprecipitation and Western blot analysis indicated co-migration of SP and FN in 27 of 31 patients with BM fibrosis. Computer-assisted molecular modeling suggested that similar secondary structural features between FN and NK-1 and the relative electrostatic charge might explain a complex formed between FN (negative) and SP (positive). This study suggests that SP may be implicated in the pathophysiology of myelofibrosis, though its role would have to be substantiated in future research.

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Active zone at Aplysia synapses: organization of presynaptic dense projections.

Journal of Neurophysiology ◽

10.1152/jn.1981.46.2.356 ◽

1981 ◽

Vol 46 (2) ◽

pp. 356-368 ◽

Cited By ~ 17

Author(s):

C H Bailey ◽

P Kandel ◽

M Chen

Keyword(s):

Active Zone ◽

Dense Projections

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Maturation of active zone assembly by Drosophila Bruchpilot

The Journal of Cell Biology ◽

10.1083/jcb.200812150 ◽

2009 ◽

Vol 186 (1) ◽

pp. 129-145 ◽

Cited By ~ 242

Author(s):

Wernher Fouquet ◽

David Owald ◽

Carolin Wichmann ◽

Sara Mertel ◽

Harald Depner ◽

...

Keyword(s):

Electron Microscopy ◽

Drosophila Melanogaster ◽

High Resolution ◽

Active Zone ◽

Synaptic Vesicles ◽

C Terminus ◽

Family Protein ◽

N Terminus ◽

Dense Projections ◽

Ca2 Channels

Synaptic vesicles fuse at active zone (AZ) membranes where Ca2+ channels are clustered and that are typically decorated by electron-dense projections. Recently, mutants of the Drosophila melanogaster ERC/CAST family protein Bruchpilot (BRP) were shown to lack dense projections (T-bars) and to suffer from Ca2+ channel–clustering defects. In this study, we used high resolution light microscopy, electron microscopy, and intravital imaging to analyze the function of BRP in AZ assembly. Consistent with truncated BRP variants forming shortened T-bars, we identify BRP as a direct T-bar component at the AZ center with its N terminus closer to the AZ membrane than its C terminus. In contrast, Drosophila Liprin-α, another AZ-organizing protein, precedes BRP during the assembly of newly forming AZs by several hours and surrounds the AZ center in few discrete punctae. BRP seems responsible for effectively clustering Ca2+ channels beneath the T-bar density late in a protracted AZ formation process, potentially through a direct molecular interaction with intracellular Ca2+ channel domains.

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DISCA: high-throughput cryo-ET structural pattern mining by deep unsupervised clustering

10.1101/2021.05.16.444381 ◽

2021 ◽

Author(s):

Xiangrui Zeng ◽

Anson Kahng ◽

Liang Xue ◽

Julia Mahamid ◽

Yi-Wei Chang ◽

...

Keyword(s):

High Throughput ◽

Pattern Mining ◽

Electron Tomography ◽

Structural Features ◽

Computer Assisted ◽

Label Free ◽

Structural Pattern ◽

Cryo Electron Tomography ◽

Homogeneous Structures

Cryo-electron tomography directly visualizes heterogeneous macromolecular structures in complex cellular environments, but existing computer-assisted sorting approaches are low-throughput or inherently limited due to their dependency on available templates and manual labels. We introduce a high-throughput template-and-label-free deep learning approach that automatically discovers subsets of homogeneous structures by learning and modeling 3D structural features and their distributions. Diverse structures emerging from sorted subsets enable systematic unbiased recognition of macromolecular complexes in situ.

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Determinants of sperm quality and fertility in domestic species

Reproduction ◽

10.1530/rep-07-0046 ◽

2007 ◽

Vol 134 (1) ◽

pp. 3-17 ◽

Cited By ~ 87

Author(s):

A M Petrunkina ◽

D Waberski ◽

A R Günzel-Apel ◽

E Töpfer-Petersen

Keyword(s):

Flow Cytometry ◽

Sperm Quality ◽

Absolute Number ◽

Fertilization Success ◽

Cell Counting ◽

Computer Assisted ◽

Timely Manner ◽

Domestic Species ◽

Image Area

Fertilization success cannot be attributed solely to the absolute number of vital, motile, morphologically normal spermatozoa inseminated into the female but more especially to their functional competence. A range ofin vitrotests has therefore been developed to monitor crucial aspects of sperm function: their ability to adapt to changing osmotic conditions, to bind to the oviductal epithelium, and to undergo capacitation in an appropriate and timely manner. The tests employ flow cytometry in conjunction with fluorescent techniques, electronic cell counting, and computer-assisted image area analysis. The highly quantitative analysis provided by electronic sizing and flow cytometry enables assessment of representative cell numbers in a very short time with high reproducibility. More importantly, it allows the detection of physiological heterogeneity within an ejaculate in terms of the development of cell subpopulations and enables the kinetic analysis of changes in living cell suspensions. The tests offer a promising strategy for evaluating fertility in domestic animals. The capability for volume regulation ensures that sperm recover from the tonic shocks experienced at ejaculation and during cryopreservation. Assessment of capacitationin vitroprovides valuable information on both the sperm’s ability to respond to fertilizing conditions and the sequence and rates of ongoing capacitation/destabilization processes. The monitoring of response to capacitating conditions in kinetic terms allows the sensitive and adequate detection of sperm populations expressing fertilization attributes and their ability to respond to external stimuli in a timely manner. However, subfertility is likely to be associated with a suboptimal response (i.e. too high or too low) rather than a minimal response.

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A presynaptic spectrin network controls active zone assembly and neurotransmitter release

10.1101/812032 ◽

2019 ◽

Author(s):

Qi Wang ◽

Lindsey Friend ◽

Rosario Vicidomini ◽

Tae Hee Han ◽

Peter Nguyen ◽

...

Keyword(s):

Active Zone ◽

Neurotransmitter Release ◽

Synaptic Cleft ◽

Structure And Function ◽

Dynamic Changes ◽

Synaptic Structure ◽

Synaptic Boutons ◽

Active Zones ◽

And Function ◽

Spectrin Network

ABSTRACTWe have previously reported that Drosophila Tenectin (Tnc) recruits αPS2/βPS integrin to ensure structural and functional integrity at larval NMJs (Wang et al., 2018). In muscles, Tnc/integrin engages the spectrin network to regulate the size and architecture of synaptic boutons. In neurons, Tnc/integrin controls neurotransmitter release. Here we show that presynaptic Tnc/integrin modulates the synaptic accumulation of key active zone components, including the Ca2+ channel Cac and the active zone scaffold Brp. Presynaptic α-Spectrin appears to be both required and sufficient for the recruitment of Cac and Brp. We visualized the endogenous α-Spectrin and found that Tnc controls spectrin recruitment at synaptic terminals. Thus, Tnc/integrin anchors the presynaptic spectrin network and ensures the proper assembly and function of the active zones. Since pre- and postsynaptic Tnc/integrin limit each other, we hypothesize that this pathway links dynamic changes within the synaptic cleft to changes in synaptic structure and function.

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Altered synaptic ultrastructure in the prefrontal cortex of Shank3-deficient rats

10.21203/rs.2.20698/v2 ◽

2020 ◽

Author(s):

Sarah Jacot-Descombes ◽

Neha U Keshav ◽

Dara L. Dickstein ◽

Bridget Wicinski ◽

William G. M. Janssen ◽

...

Keyword(s):

Prefrontal Cortex ◽

Rat Model ◽

Preliminary Evidence ◽

Autism Spectrum ◽

Dendritic Morphology ◽

Scaffolding Protein ◽

Spine Density ◽

Synaptic Structure ◽

Synaptic Ultrastructure ◽

Behavioral Impairments

Abstract Background Deletion or mutations of SHANK3 lead to Phelan-McDermid syndrome and monogenic forms of autism spectrum disorder (ASD). SHANK3 encodes its eponymous scaffolding protein at excitatory glutamatergic synapses. Altered morphology of dendrites and spines in the hippocampus, cerebellum, and striatum have been associated with behavioral impairments in Shank3-deficient animal models. Given the attentional deficit in these animals, our study explored whether deficiency of Shank3 in a rat model alters neuron morphology and synaptic ultrastructure in the medial prefrontal cortex (mPFC). Methods We assessed dendrite and spine morphology and spine density in mPFC layer III neurons in Shank3 -homozygous knockout ( Shank3 -KO), heterozygous ( Shank3 -Het), and wild-type (WT) rats. We used electron microscopy to determine the density of asymmetric synapses in mPFC layer III excitatory neurons in these rats. We measured postsynaptic density (PSD) length, PSD area, and head diameter (HD) of spines at these synapses. Results Basal dendritic morphology was similar among the three genotypes. Spine density and morphology were comparable, but more thin and mushroom spines had larger head volumes in Shank3 -Het compared to WT and Shank3 -KO. All three groups had comparable synapse density and PSD length. Spine HD of total and non-perforated synapses in Shank3 -Het rats, but not Shank3 -KO rats, was significantly larger than in WT rats. The total and non-perforated PSD area was significantly larger in Shank3 -Het rats compared to Shank3 -KO rats. These findings represent preliminary evidence for synaptic ultrastructural alterations in the mPFC of rats that lack one copy of Shank3 and mimic the heterozygous loss of SHANK3 in Phelan-McDermid syndrome. Limitations The Shank3 deletion in the rat model we used does not affect all isoforms of the protein and would only model the effect of the mutations resulting in loss of the N-terminus of the protein. Given the higher prevalence of ASD in males, the ultrastructural study focused only on synaptic structure in male Shank3 -deficient rats. Conclusions We observed increased HD and PSD area in Shank3 -Het rats. These observations suggest the occurrence of altered synaptic ultrastructure in this animal model, further pointing to a key role of defective expression of the Shank3 protein in ASD and Phelan-McDermid syndrome.

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Computational Modulation of the V3 Region of Glycoprotein gp125 of HIV-2

International Journal of Molecular Sciences ◽

10.3390/ijms22041948 ◽

2021 ◽

Vol 22 (4) ◽

pp. 1948

Author(s):

Patrícia A. Serra ◽

Nuno Taveira ◽

Rita C. Guedes

Keyword(s):

Viral Entry ◽

Network Formation ◽

Protein Structures ◽

3D Structure ◽

3D Models ◽

Structural Features ◽

Host Cells ◽

Computer Assisted ◽

Function Relationship ◽

Dynamics Simulations

HIV-2 infection is frequently neglected in HIV/AIDS campaigns. However, a special emphasis must be given to HIV-2 as an untreated infection that also leads to AIDS and death, and for which the efficacy of most available drugs is limited against HIV-2. HIV envelope glycoproteins mediate binding to the receptor CD4 and co-receptors at the surface of the target cell, enabling fusion with the cell membrane and viral entry. Here, we developed and optimized a computer-assisted drug design approach of an important HIV-2 glycoprotein that allows us to explore and gain further insights at the molecular level into protein structures and interactions crucial for the inhibition of HIV-2 cell entry. The 3D structure of a key HIV-2ROD gp125 region was generated by a homology modeling campaign. To disclose the importance of the main structural features and compare them with experimental results, 3D-models of six mutants were also generated. These mutations revealed the selective impact on the behavior of the protein. Furthermore, molecular dynamics simulations were performed to optimize the models, and the dynamic behavior was tackled to account for structure flexibility and interactions network formation. Structurally, the mutations studied lead to a loss of aromatic features, which is very important for the establishment of π-π interactions and could induce a structural preference by a specific coreceptor. These new insights into the structure-function relationship of HIV-2 gp125 V3 and surrounding regions will help in the design of better models and the design of new small molecules capable to inhibit the attachment and binding of HIV with host cells.

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Altered synaptic ultrastructure in the prefrontal cortex of Shank3-deficient rats

Molecular Autism ◽

10.1186/s13229-020-00393-8 ◽

2020 ◽

Vol 11 (1) ◽

Author(s):

Sarah Jacot-Descombes ◽

Neha U. Keshav ◽

Dara L. Dickstein ◽

Bridget Wicinski ◽

William G. M. Janssen ◽

...

Keyword(s):

Prefrontal Cortex ◽

Rat Model ◽

Preliminary Evidence ◽

Autism Spectrum ◽

Dendritic Morphology ◽

Scaffolding Protein ◽

Spine Density ◽

Synaptic Structure ◽

Synaptic Ultrastructure ◽

Behavioral Impairments

Abstract Background Deletion or mutations of SHANK3 lead to Phelan–McDermid syndrome and monogenic forms of autism spectrum disorder (ASD). SHANK3 encodes its eponymous scaffolding protein at excitatory glutamatergic synapses. Altered morphology of dendrites and spines in the hippocampus, cerebellum, and striatum have been associated with behavioral impairments in Shank3-deficient animal models. Given the attentional deficit in these animals, our study explored whether deficiency of Shank3 in a rat model alters neuron morphology and synaptic ultrastructure in the medial prefrontal cortex (mPFC). Methods We assessed dendrite and spine morphology and spine density in mPFC layer III neurons in Shank3-homozygous knockout (Shank3-KO), heterozygous (Shank3-Het), and wild-type (WT) rats. We used electron microscopy to determine the density of asymmetric synapses in mPFC layer III excitatory neurons in these rats. We measured postsynaptic density (PSD) length, PSD area, and head diameter (HD) of spines at these synapses. Results Basal dendritic morphology was similar among the three genotypes. Spine density and morphology were comparable, but more thin and mushroom spines had larger head volumes in Shank3-Het compared to WT and Shank3-KO. All three groups had comparable synapse density and PSD length. Spine HD of total and non-perforated synapses in Shank3-Het rats, but not Shank3-KO rats, was significantly larger than in WT rats. The total and non-perforated PSD area was significantly larger in Shank3-Het rats compared to Shank3-KO rats. These findings represent preliminary evidence for synaptic ultrastructural alterations in the mPFC of rats that lack one copy of Shank3 and mimic the heterozygous loss of SHANK3 in Phelan–McDermid syndrome. Limitations The Shank3 deletion in the rat model we used does not affect all isoforms of the protein and would only model the effect of mutations resulting in loss of the N-terminus of the protein. Given the higher prevalence of ASD in males, the ultrastructural study focused only on synaptic structure in male Shank3-deficient rats. Conclusions We observed increased HD and PSD area in Shank3-Het rats. These observations suggest the occurrence of altered synaptic ultrastructure in this animal model, further pointing to a key role of defective expression of the Shank3 protein in ASD and Phelan–McDermid syndrome.

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