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Unraveling the Web of Challenges Around Understanding How Black Widow Spiders Spin Silk /

Record Type:	Electronic resources : Monograph/item
Title/Author:	Unraveling the Web of Challenges Around Understanding How Black Widow Spiders Spin Silk // Christopher A. L Sharpe.
Author:	Sharpe, Christopher A. L.,
Description:	1 electronic resource (228 pages)
Notes:	Source: Dissertations Abstracts International, Volume: 86-12, Section: B.
Contained By:	Dissertations Abstracts International86-12B.
Subject:	Materials science. -
Online resource:	https://pqdd.sinica.edu.tw/twdaoapp/servlet/advanced?query=31933616
ISBN:	9798315799566

Unraveling the Web of Challenges Around Understanding How Black Widow Spiders Spin Silk /
Sharpe, Christopher A. L.,

Unraveling the Web of Challenges Around Understanding How Black Widow Spiders Spin Silk /Christopher A. L Sharpe. - 1 electronic resource (228 pages)

Source: Dissertations Abstracts International, Volume: 86-12, Section: B.

Producing synthetic spider silks has been a Holy Grail of materials science for centuries, but only relatively recently have we started to make significant progress towards that goal. Only in the last few decades have we discovered the ingredients to the spider silk recipe - the protein composition, sequences, and concentration - and the general steps that occur during spinning: changes in pH, ion exchange, dehydration, and shear. However, we still lack a good understanding of what these processing steps do to the silk proteins themselves and how they give rise to the final, complex fiber. Such an understanding is critical to transferring the in vivo system into an industrial process capable of producing large quantities of high performance silk, especially since the synthesis process for synthetic vs natural silks is so different. Special techniques need to be employed to "peak under the hood" and see how the silk proteins evolve during the spinning process. Due to native silk's high concentration and extreme sensitivity to multiple stimuli, only a handful of techniques can be used to probe its structure. In this dissertation, two techniques have been employed: 1) traditional Cryogenic Transmission Electron Microscopy (Cryo-TEM), and 2) High Pressure Freezing - Freeze Substitution (HPF-FS) ultrastructure electron microscopy. Cryo-TEM uses very dilute concentrations of sample, which must be prepared carefully to minimize shear and chemical effects; however, this dilute state is much closer to its true, hydrated state than most other techniques permit. HPF-FS allows for direct imaging of the in vivo tissue and observation of the native structural thanks to flash-freezing the entire silk gland at high pressure to preserve native structures and prevent many artifact-forming processes, followed by fixing and staining at cryogenic temperatures in organic solvent. This process prevents many of the artifacts found in chemical fixation, as the cryogenic temperatures of freeze substitution minimize many osmotic effects. This is key for preserving the silk, as pH and dehydration effects are known stimuli in its spinning process. HPF-FS also incorporates metal stains for EM contrast and concludes with embedding the sample into epoxy resin, protecting it from most shear effects as well. By avoiding stimuli that trigger the silk protein assembly process, HPF-FS preserves the native silk gland structures. The epoxy-embedded sample "chits" are then sectioned with a diamond knife and imaged by SEM or TEM.However, HPF is normally constrained to samples less than 200 μm thick, while the silk glands are 400+ μm thick - introducing a potentially fatal problem. For samples thicker than 200 μm, the high pressure is almost universally insufficient to prevent the formation of large, tissue-damaging ice crystals that alter the native structure. To characterize ice crystallite size, I employed Cryogenic Wide Angle X-ray Scattering (Cryo-WAXS) for the post-HPF pre-FS silk glands. This showed that most HPF samples had ice crystallites smaller than previously-characterized silk micelles - clearing the way for ultrastructure characterization, but raising questions as to why the silk glands were able to undergo HPF so well. To date, only cryoprotectant-infiltrated or inherently cryoprotective thick samples have been able to go through HPF without significant ice damage. We tested the Ice Recrystallization Inhibition (IRI) activity of black widow dragline silk, and found it to have an IRI IC50 of 0.14±0.03 mg/mL. Compared to the native silk concentration of ~400 mg/mL, this essentially ensures that the silk itself confers the cryoprotective effect enabling successful HPF. However, whether that activity is due to particular proteins in the dragline silk, particular conformations they exhibit prior to spinning, or another factor still needs to be explored.Finally, I describe a workflow for how to reconstruct the entirety of a silk gland at nanoscale resolution. This reconstruction process, while not complete, hopefully will serve as a guide to others undertaking tissue reconstruction projects of this scale.

English

ISBN: 9798315799566Subjects--Topical Terms:

543314
Materials science.
Subjects--Index Terms:

Black widow

Unraveling the Web of Challenges Around Understanding How Black Widow Spiders Spin Silk /
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520 $a Producing synthetic spider silks has been a Holy Grail of materials science for centuries, but only relatively recently have we started to make significant progress towards that goal. Only in the last few decades have we discovered the ingredients to the spider silk recipe - the protein composition, sequences, and concentration - and the general steps that occur during spinning: changes in pH, ion exchange, dehydration, and shear. However, we still lack a good understanding of what these processing steps do to the silk proteins themselves and how they give rise to the final, complex fiber. Such an understanding is critical to transferring the in vivo system into an industrial process capable of producing large quantities of high performance silk, especially since the synthesis process for synthetic vs natural silks is so different. Special techniques need to be employed to "peak under the hood" and see how the silk proteins evolve during the spinning process. Due to native silk's high concentration and extreme sensitivity to multiple stimuli, only a handful of techniques can be used to probe its structure. In this dissertation, two techniques have been employed: 1) traditional Cryogenic Transmission Electron Microscopy (Cryo-TEM), and 2) High Pressure Freezing - Freeze Substitution (HPF-FS) ultrastructure electron microscopy. Cryo-TEM uses very dilute concentrations of sample, which must be prepared carefully to minimize shear and chemical effects; however, this dilute state is much closer to its true, hydrated state than most other techniques permit. HPF-FS allows for direct imaging of the in vivo tissue and observation of the native structural thanks to flash-freezing the entire silk gland at high pressure to preserve native structures and prevent many artifact-forming processes, followed by fixing and staining at cryogenic temperatures in organic solvent. This process prevents many of the artifacts found in chemical fixation, as the cryogenic temperatures of freeze substitution minimize many osmotic effects. This is key for preserving the silk, as pH and dehydration effects are known stimuli in its spinning process. HPF-FS also incorporates metal stains for EM contrast and concludes with embedding the sample into epoxy resin, protecting it from most shear effects as well. By avoiding stimuli that trigger the silk protein assembly process, HPF-FS preserves the native silk gland structures. The epoxy-embedded sample "chits" are then sectioned with a diamond knife and imaged by SEM or TEM.However, HPF is normally constrained to samples less than 200 μm thick, while the silk glands are 400+ μm thick - introducing a potentially fatal problem. For samples thicker than 200 μm, the high pressure is almost universally insufficient to prevent the formation of large, tissue-damaging ice crystals that alter the native structure. To characterize ice crystallite size, I employed Cryogenic Wide Angle X-ray Scattering (Cryo-WAXS) for the post-HPF pre-FS silk glands. This showed that most HPF samples had ice crystallites smaller than previously-characterized silk micelles - clearing the way for ultrastructure characterization, but raising questions as to why the silk glands were able to undergo HPF so well. To date, only cryoprotectant-infiltrated or inherently cryoprotective thick samples have been able to go through HPF without significant ice damage. We tested the Ice Recrystallization Inhibition (IRI) activity of black widow dragline silk, and found it to have an IRI IC50 of 0.14±0.03 mg/mL. Compared to the native silk concentration of ~400 mg/mL, this essentially ensures that the silk itself confers the cryoprotective effect enabling successful HPF. However, whether that activity is due to particular proteins in the dragline silk, particular conformations they exhibit prior to spinning, or another factor still needs to be explored.Finally, I describe a workflow for how to reconstruct the entirety of a silk gland at nanoscale resolution. This reconstruction process, while not complete, hopefully will serve as a guide to others undertaking tissue reconstruction projects of this scale.
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653 $a Black widow
653 $a Cryo-TEM
653 $a Cryo-WAXS
653 $a Cryoprotectant
653 $a Spider silk
653 $a Ultrastructure
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710 2 $a Northwestern University. $b Materials Science and Engineering. $e degree granting institution. $3 3817073
720 1 $a Gianneschi, Nathan C. $e degree supervisor.
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