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035 a| (OCoLC)1450451809;

096 a| QU 375;

049 a| EREE;

100 1 a| Czika, Wesley Cole,e| author.;

245 10 a| FRET not, shedding light on myosin mechanical forces /c| by Wesley Cole Czika.;

264 1 a| [Greenville, N.C.] :b| [East Carolina University],c| 2024.;

300 a| 1 online resource (79 pages) :b| illustrations (chiefly color);

336 a| textb| txt2| rdacontent;

337 a| computerb| c2| rdamedia;

338 a| online resourceb| cr2| rdacarrier;

347 a| text fileb| application/pdfc| 2.8 MB2| rda;

490 1 a| ECU Brody School of Medicine thesis;

538 a| System requirements: Adobe Reader.;

538 a| Mode of access: World Wide Web.;

502 b| M.S.c| East Carolina Universityd| 2024.;

500 a| Presented to the Faculty of the Department of Anatomy and Cell Biology;

500 a| Advisor: Karen Litwa;

500 a| Title from PDF t.p. (viewed October 3, 2025).;

520 3 a| Mechanotransduction, the conversion of mechanical stimuli into biochemical signals, is crucial for cellular function. Intracellularly, rearrangements of the actin cytoskeleton mediate force production across adhesions, leading to changes in adhesion composition and signaling. The actin-associated motor protein, Non-Muscle Myosin II (NMII) generates forces by contracting actin filaments. In the brain, synapses are adhesive sites that mediate electrochemical signaling between neurons. Post-synaptic dendritic spines are actin-enriched structures that specifically contain the Non-Muscle Myosin IIB isoform (NMIIB). NMIIB activity is necessary and sufficient to drive dendritic spine maturation. However, it is unclear whether NMIIB activity generates synaptic forces, in part due to the lack of suitable tools to measure microscale-localized forces within spines. The recent development of FRET-based tension sensors allows forces to be measured with spatial and temporal precision. Using NMIIB engineered to contain a FRET tension sensor, we were able to assess forces associated with synapse development. In vitro, mouse neurons exhibit a well-characterized timeline of synapse development, with synapse formation around 7DIV and maturation occurring between 14-21DIV. Using the NMIIB FRET tension sensor, we observed increased forces at 7DIV that were significantly reduced at 14DIV, suggesting that NMIIB-mediated forces are highest during synapse formation. Inhibition of NMIIB activity with the RhoA kinase inhibitor, Y-27632, prevented force-dependent changes in FRET, demonstrating that the observed changes are myosin-dependent. We similarly translated these findings into two independent hIPSC-derived neuron lines, in which we observed synaptic forces after 7 days of neural differentiation that were NMIIB-dependent. In addition to reducing NMIIB force production, pharmacological inhibition increased the formation of dendritic spine precursors. Furthermore, these spine precursors were significantly longer than those found in the control untreated neurons. This study is the first demonstration of synaptic mechanotransduction. Our findings identify a key developmental window associated with synapse formation in which NMIIB-mediated forces are highest. Furthermore, we observed developmental conservation of NMIIB-mediated synaptic force production between rodent and human neurons. Understanding how forces orchestrate synaptic scaffolds in development will likely have implications for the numerous neurodevelopmental disorders associated with altered actomyosin regulation.;

504 a| Includes bibliographical references.;

650 0 a| Myosin.;

650 2 a| Mechanotransduction, Cellular0| (DNLM)D040542;

650 2 a| Fluorescence Resonance Energy Transfer0| (DNLM)D031541;

650 2 a| Myosins0| (DNLM)D009218;

650 2 a| Mechanical Phenomena0| (DNLM)D055595;

653 a| Non-Muscle Myosin II;

653 a| Dendritic Spines;

653 a| Actin Cytoskeleton;

653 a| FRET;

700 1 a| Litwa, Karen,e| degree supervisor.;

710 2 a| East Carolina University.b| Department of Anatomy and Cell Biology.;

830 0 a| ECU Brody School of Medicine thesis.;

856 40 z| Access via ScholarShipu| http://hdl.handle.net/10342/13515;

994 a| C0b| ERE;

596 a| 1 4;

998 a| 6462809;

999 a| CLICK ON WEB ADDRESSw| ASISc| 1i| 6462809-1001l| JNETm| JOYNERr| Ys| Yt| JNE3ETDu| 7/31/2024x| ETDz| JERESOURCE;

999 a| CLICK ON WEB ADDRESSw| ASISc| 1i| 6462809-2001l| HSLELECm| HSLr| Ys| Yt| HEETDu| 7/31/2024x| ETDz| HERESOURCE;

FRET not, shedding light on myosin mechanical forces / by Wesley Cole Czika.

Author/creator	Czika, Wesley Cole author.
Other author	Litwa, Karen, degree supervisor.
Other author	East Carolina University. Department of Anatomy and Cell Biology.
Format	Theses and dissertations
Publication	[Greenville, N.C.] : [East Carolina University], 2024.
Description	1 online resource (79 pages) : illustrations (chiefly color)
Supplemental Content	Access via ScholarShip
Subjects	Myosin. Mechanotransduction, Cellular Fluorescence Resonance Energy Transfer Myosins Mechanical Phenomena

Series	ECU Brody School of Medicine thesis ECU Brody School of Medicine thesis.
Summary	Mechanotransduction, the conversion of mechanical stimuli into biochemical signals, is crucial for cellular function. Intracellularly, rearrangements of the actin cytoskeleton mediate force production across adhesions, leading to changes in adhesion composition and signaling. The actin-associated motor protein, Non-Muscle Myosin II (NMII) generates forces by contracting actin filaments. In the brain, synapses are adhesive sites that mediate electrochemical signaling between neurons. Post-synaptic dendritic spines are actin-enriched structures that specifically contain the Non-Muscle Myosin IIB isoform (NMIIB). NMIIB activity is necessary and sufficient to drive dendritic spine maturation. However, it is unclear whether NMIIB activity generates synaptic forces, in part due to the lack of suitable tools to measure microscale-localized forces within spines. The recent development of FRET-based tension sensors allows forces to be measured with spatial and temporal precision. Using NMIIB engineered to contain a FRET tension sensor, we were able to assess forces associated with synapse development. In vitro, mouse neurons exhibit a well-characterized timeline of synapse development, with synapse formation around 7DIV and maturation occurring between 14-21DIV. Using the NMIIB FRET tension sensor, we observed increased forces at 7DIV that were significantly reduced at 14DIV, suggesting that NMIIB-mediated forces are highest during synapse formation. Inhibition of NMIIB activity with the RhoA kinase inhibitor, Y-27632, prevented force-dependent changes in FRET, demonstrating that the observed changes are myosin-dependent. We similarly translated these findings into two independent hIPSC-derived neuron lines, in which we observed synaptic forces after 7 days of neural differentiation that were NMIIB-dependent. In addition to reducing NMIIB force production, pharmacological inhibition increased the formation of dendritic spine precursors. Furthermore, these spine precursors were significantly longer than those found in the control untreated neurons. This study is the first demonstration of synaptic mechanotransduction. Our findings identify a key developmental window associated with synapse formation in which NMIIB-mediated forces are highest. Furthermore, we observed developmental conservation of NMIIB-mediated synaptic force production between rodent and human neurons. Understanding how forces orchestrate synaptic scaffolds in development will likely have implications for the numerous neurodevelopmental disorders associated with altered actomyosin regulation.
General note	Presented to the Faculty of the Department of Anatomy and Cell Biology
General note	Advisor: Karen Litwa
General note	Title from PDF t.p. (viewed October 3, 2025).
Dissertation note	M.S. East Carolina University 2024.
Bibliography note	Includes bibliographical references.
Technical details	System requirements: Adobe Reader.
Technical details	Mode of access: World Wide Web.

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FRET not, shedding light on myosin mechanical forces / by Wesley Cole Czika.

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