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Article ; Online: A Step-by-Step Guide to Instant Structured Illumination Microscopy (iSIM).

Zhovmer, Alexander / Combs, Christian A

Methods in molecular biology (Clifton, N.J.)

2021 Volume 2304, Page(s) 347–359

Abstract: Instant structured illumination microscopy (iSIM) allows for rapid multicolor three-dimensional fluorescence imaging at levels of resolution approaching twice the diffraction limit. Here we briefly describe the theory of iSIM and outline a typical ... ...

Abstract	Instant structured illumination microscopy (iSIM) allows for rapid multicolor three-dimensional fluorescence imaging at levels of resolution approaching twice the diffraction limit. Here we briefly describe the theory of iSIM and outline a typical hardware setup. We also provide step-by-step guides for generating a cellular-based fluorescent standard, obtaining a multicolor image with iSIM, and the post-processing steps of de-striping and deconvolution using freely distributed software to minimize time and expense. A "Notes" section is also given to inform the reader of the limitations and considerations for the methods shown. Also discussed are alternative methods, quality control checks, and considerations for two-camera alignment.
MeSH term(s)	Animals ; Cell Line ; Fibroblasts/cytology ; Imaging, Three-Dimensional/instrumentation ; Mice ; Microscopy, Fluorescence/instrumentation ; Software
Language	English
Publishing date	2021-05-24
Publishing country	United States
Document type	Journal Article ; Research Support, N.I.H., Intramural
ISSN	1940-6029
ISSN (online)	1940-6029
DOI	10.1007/978-1-0716-1402-0_19
Database	MEDical Literature Analysis and Retrieval System OnLINE

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Article ; Online: Septins provide microenvironment sensing and cortical actomyosin partitioning in motile amoeboid T lymphocytes.

Zhovmer, Alexander S / Manning, Alexis / Smith, Chynna / Nguyen, Ashley / Prince, Olivia / Sáez, Pablo J / Ma, Xuefei / Tsygankov, Denis / Cartagena-Rivera, Alexander X / Singh, Niloy A / Singh, Rakesh K / Tabdanov, Erdem D

Science advances

2024 Volume 10, Issue 1, Page(s) eadi1788

Abstract: The all-terrain motility of lymphocytes in tissues and tissue-like gels is best described as amoeboid motility. For amoeboid motility, lymphocytes do not require specific biochemical or structural modifications to the surrounding extracellular matrix. ... ...

Abstract	The all-terrain motility of lymphocytes in tissues and tissue-like gels is best described as amoeboid motility. For amoeboid motility, lymphocytes do not require specific biochemical or structural modifications to the surrounding extracellular matrix. Instead, they rely on changing shape and steric interactions with the microenvironment. However, the exact mechanism of amoeboid motility remains elusive. Here, we report that septins participate in amoeboid motility of T cells, enabling the formation of F-actin and α-actinin-rich cortical rings at the sites of cell cortex-indenting collisions with the extracellular matrix. Cortical rings compartmentalize cells into chains of spherical segments that are spatially conformed to the available lumens, forming transient "hourglass"-shaped steric locks onto the surrounding collagen fibers. The steric lock facilitates pressure-driven peristaltic propulsion of cytosolic content by individually contracting cell segments. Our results suggest that septins provide microenvironment-guided partitioning of actomyosin contractility and steric pivots required for amoeboid motility of T cells in tissue-like microenvironments.
MeSH term(s)	Actomyosin/metabolism ; Septins/metabolism ; Cell Movement ; Amoeba/metabolism ; T-Lymphocytes/metabolism
Chemical Substances	Actomyosin (9013-26-7) ; Septins (EC 3.6.1.-)
Language	English
Publishing date	2024-01-03
Publishing country	United States
Document type	Journal Article
ZDB-ID	2810933-8
ISSN	2375-2548 ; 2375-2548
ISSN (online)	2375-2548
ISSN	2375-2548
DOI	10.1126/sciadv.adi1788
Database	MEDical Literature Analysis and Retrieval System OnLINE

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Article ; Online: Engineering Elastic Nano- and Micro-Patterns and Textures for Directed Cell Motility.

Tabdanov, Erdem D / Zhovmer, Alexander S / Puram, Vikram / Provenzano, Paolo P

STAR protocols

2020 Volume 1, Issue 1

Abstract: We present a reproducible protocol for fabrication of polyacrylamide (PAA) hydrogel-based nano-patterns and nano-textures with a wide range of elastic rigidities to study fundamental cell behaviors, such as mechanosensitivity and motility. We explore the ...

Abstract	We present a reproducible protocol for fabrication of polyacrylamide (PAA) hydrogel-based nano-patterns and nano-textures with a wide range of elastic rigidities to study fundamental cell behaviors, such as mechanosensitivity and motility. We explore the benefits of this protocol by successfully testing the compatibility of the PAA platforms with super-resolution microscopy, which is largely unavailable with platforms of nano-scale textures made from different polymers. We also utilized soft and rigid nano-textures to study the mechanosensing basis of T cell behavior and phenotype. For complete information on the generation and use of this protocol, please refer to Tabdanov et al. (2018b).
MeSH term(s)	Acrylic Resins ; Cell Engineering/methods ; Cell Line, Tumor ; Cell Movement ; Humans ; Nanotechnology/methods
Chemical Substances	Acrylic Resins ; polyacrylamide gels
Language	English
Publishing date	2020-06-03
Publishing country	United States
Document type	Journal Article ; Research Support, N.I.H., Extramural ; Research Support, N.I.H., Intramural ; Research Support, Non-U.S. Gov't ; Research Support, U.S. Gov't, Non-P.H.S.
ISSN	2666-1667
ISSN (online)	2666-1667
DOI	10.1016/j.xpro.2019.100013
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Article ; Online: Proximal disruptor aided ligation (ProDAL) of kilobase-long RNAs.

Zhovmer, Alexander / Qu, Xiaohui

RNA biology

2016 Volume 13, Issue 7, Page(s) 613–621

Abstract: RNA with site-specific modification is a useful tool for RNA biology studies. However, generating kilobase (kb) -long RNA with internal modification at a site distant from RNA termini remains challenging. Here we report an enhanced splint ligation ... ...

Abstract	RNA with site-specific modification is a useful tool for RNA biology studies. However, generating kilobase (kb) -long RNA with internal modification at a site distant from RNA termini remains challenging. Here we report an enhanced splint ligation technique, proximal disruptor aided ligation (ProDAL), which allows adequate efficiency toward this purpose. The key to our approach is using multiple DNA oligonucleotides, 'proximal disruptors', to target the RNA substrate sequence next to the ligation site. The binding of disruptors helps to free the ligation site from intramolecular RNA basepairing, and consequently promotes more efficient formation of the pre-ligation complex and a higher overall ligation yield. We used naturally occurring 1.0 kb renilla and 1.9 kb firefly luciferase mRNA sequences to test the efficacy of our approach. ProDAL yielded 9-14% efficiency for the ligation between two RNA substrates, both of which were between 414 and 1313 nucleotides (nt) long. ProDAL also allowed similarly high efficiency for generating kb-long RNA with site-specific internal modification by a simple three-part ligation between two long RNA substrates and a modification-carrying RNA oligonucleotide. In comparison, classical splint ligation yielded a significantly lower efficiency of 0-2% in all cases. We expect that ProDAL will benefit studies involving kb-long RNAs, including translation, long non-coding RNAs, RNA splicing and modification, and large ribonucleoprotein complexes.
MeSH term(s)	RNA/chemical synthesis ; RNA/chemistry
Chemical Substances	RNA (63231-63-0)
Language	English
Publishing date	2016-07-02
Publishing country	United States
Document type	Journal Article
ISSN	1555-8584
ISSN (online)	1555-8584
DOI	10.1080/15476286.2016.1189072
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Article ; Online: Programmable DNA-augmented hydrogels for controlled activation of human lymphocytes.

Zhovmer, Alexander S / Chandler, Morgan / Manning, Alexis / Afonin, Kirill A / Tabdanov, Erdem D

Nanomedicine : nanotechnology, biology, and medicine

2021 Volume 37, Page(s) 102442

Abstract: Contractile forces within the planar interface between T cell and antigen-presenting surface mechanically stimulate T cell receptors (TCR) in the mature immune synapses. However, the origin of mechanical stimulation during the initial, i.e., presynaptic, ...

Abstract	Contractile forces within the planar interface between T cell and antigen-presenting surface mechanically stimulate T cell receptors (TCR) in the mature immune synapses. However, the origin of mechanical stimulation during the initial, i.e., presynaptic, microvilli-based TCR activation in the course of immune surveillance remains unknown and new tools to help address this problem are needed. In this work, we develop nucleic acid nanoassembly (NAN)-based technology for functionalization of hydrogels using isothermal toehold-mediated reassociation of RNA/DNA heteroduplexes. Resulting platform allows for regulation with NAN linkers of 3D force momentum along the TCR mechanical axis, whereas hydrogels contribute to modulation of 2D shear modulus. By utilizing different lengths of NAN linkers conjugated to polyacrylamide gels of different shear moduli, we demonstrate an efficient capture of human T lymphocytes and tunable activation of TCR, as confirmed by T-cell spreading and pY foci.
MeSH term(s)	Antigen-Presenting Cells/drug effects ; DNA/chemistry ; DNA/pharmacology ; Humans ; Hydrogels/chemistry ; Hydrogels/pharmacology ; Lymphocyte Activation/drug effects ; Lymphocyte Activation/genetics ; Lymphocytes/drug effects ; Lymphocytes/metabolism ; Nucleic Acid Heteroduplexes/chemistry ; Nucleic Acid Heteroduplexes/genetics ; Nucleic Acid Heteroduplexes/pharmacology ; RNA/chemistry ; RNA/genetics ; Receptors, Antigen, T-Cell/drug effects ; Receptors, Antigen, T-Cell/genetics ; T-Lymphocytes/drug effects
Chemical Substances	Hydrogels ; Nucleic Acid Heteroduplexes ; Receptors, Antigen, T-Cell ; RNA (63231-63-0) ; DNA (9007-49-2)
Language	English
Publishing date	2021-07-17
Publishing country	United States
Document type	Journal Article ; Research Support, N.I.H., Extramural ; Research Support, N.I.H., Intramural ; Research Support, Non-U.S. Gov't
ZDB-ID	2183417-9
ISSN	1549-9642 ; 1549-9634
ISSN (online)	1549-9642
ISSN	1549-9634
DOI	10.1016/j.nano.2021.102442
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Article: Septins Enable T Cell Contact Guidance

Zhovmer, Alexander S / Manning, Alexis / Smith, Chynna / Wang, Jian / Ma, Xuefei / Tsygankov, Denis / Dokholyan, Nikolay V / Cartagena-Rivera, Alexander X / Singh, Rakesh K / Tabdanov, Erdem D

bioRxiv : the preprint server for biology

2023

Abstract: Lymphocytes exit circulation and enter in-tissue guided migration toward sites of tissue pathologies, damage, infection, or inflammation. By continuously sensing and adapting to the guiding chemo-mechano-structural properties of the tissues, lymphocytes ... ...

Abstract	Lymphocytes exit circulation and enter in-tissue guided migration toward sites of tissue pathologies, damage, infection, or inflammation. By continuously sensing and adapting to the guiding chemo-mechano-structural properties of the tissues, lymphocytes dynamically alternate and combine their amoeboid (non-adhesive) and mesenchymal (adhesive) migration modes. However, which mechanisms guide and balance different migration modes are largely unclear. Here we report that suppression of septins GTPase activity induces an abrupt amoeboid-to-mesenchymal transition of T cell migration mode, characterized by a distinct, highly deformable integrin-dependent immune cell contact guidance. Surprisingly, the T cell actomyosin cortex contractility becomes diminished, dispensable and antagonistic to mesenchymal-like migration mode. Instead, mesenchymal-like T cells rely on microtubule stabilization and their non-canonical dynein motor activity for high fidelity contact guidance. Our results establish septin's GTPase activity as an important on/off switch for integrin-dependent migration of T lymphocytes, enabling their dynein-driven fluid-like mesenchymal propulsion along the complex adhesion cues.
Language	English
Publishing date	2023-09-27
Publishing country	United States
Document type	Preprint
DOI	10.1101/2023.09.26.559597
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Article: Dynein-Powered Cell Locomotion Guides Metastasis of Breast Cancer.

Tagay, Yerbol / Kheirabadi, Sina / Ataie, Zaman / Singh, Rakesh K / Prince, Olivia / Nguyen, Ashley / Zhovmer, Alexander S / Ma, Xuefei / Sheikhi, Amir / Tsygankov, Denis / Tabdanov, Erdem D

bioRxiv : the preprint server for biology

2023

Abstract: Metastasis is a principal cause of death in cancer patients, which remains an unresolved fundamental and clinical problem. Conventionally, metastatic dissemination is linked to the actomyosin-driven cell locomotion. However, locomotion of cancer cells ... ...

Abstract	Metastasis is a principal cause of death in cancer patients, which remains an unresolved fundamental and clinical problem. Conventionally, metastatic dissemination is linked to the actomyosin-driven cell locomotion. However, locomotion of cancer cells often does not strictly line up with the measured actomyosin forces. Here, we identify a complementary mechanism of metastatic locomotion powered by the dynein-generated forces. These forces that arise within a non-stretchable microtubule network drive persistent contact guidance of migrating cancer cells along the biomimetic collagen fibers. We also show that dynein-powered locomotion becomes indispensable during invasive 3D migration within a tissue-like luminal network between spatially confining hydrogel microspheres. Our results indicate that the complementary contractile system of dynein motors and microtubules is always necessary and in certain instances completely sufficient for dissemination of metastatic breast cancer cells. These findings advance fundamental understanding of cell locomotion mechanisms and expand the spectrum of clinical targets against metastasis.
Language	English
Publishing date	2023-04-05
Publishing country	United States
Document type	Preprint
DOI	10.1101/2023.04.04.535605
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Article ; Online: Dynein-Powered Cell Locomotion Guides Metastasis of Breast Cancer.

Tagay, Yerbol / Kheirabadi, Sina / Ataie, Zaman / Singh, Rakesh K / Prince, Olivia / Nguyen, Ashley / Zhovmer, Alexander S / Ma, Xuefei / Sheikhi, Amir / Tsygankov, Denis / Tabdanov, Erdem D

Advanced science (Weinheim, Baden-Wurttemberg, Germany)

2023 Volume 10, Issue 31, Page(s) e2302229

Abstract: The principal cause of death in cancer patients is metastasis, which remains an unresolved problem. Conventionally, metastatic dissemination is linked to actomyosin-driven cell locomotion. However, the locomotion of cancer cells often does not strictly ... ...

Abstract	The principal cause of death in cancer patients is metastasis, which remains an unresolved problem. Conventionally, metastatic dissemination is linked to actomyosin-driven cell locomotion. However, the locomotion of cancer cells often does not strictly line up with the measured actomyosin forces. Here, a complementary mechanism of metastatic locomotion powered by dynein-generated forces is identified. These forces arise within a non-stretchable microtubule network and drive persistent contact guidance of migrating cancer cells along the biomimetic collagen fibers. It is also shown that the dynein-powered locomotion becomes indispensable during invasive 3D migration within a tissue-like luminal network formed by spatially confining granular hydrogel scaffolds (GHS) made up of microscale hydrogel particles (microgels). These results indicate that the complementary motricity mediated by dynein is always necessary and, in certain instances, sufficient for disseminating metastatic breast cancer cells. These findings advance the fundamental understanding of cell locomotion mechanisms and expand the spectrum of clinical targets against metastasis.
MeSH term(s)	Humans ; Female ; Dyneins/metabolism ; Breast Neoplasms ; Actomyosin/metabolism ; Cell Movement ; Hydrogels
Chemical Substances	Dyneins (EC 3.6.4.2) ; Actomyosin (9013-26-7) ; Hydrogels
Language	English
Publishing date	2023-09-19
Publishing country	Germany
Document type	Journal Article
ZDB-ID	2808093-2
ISSN	2198-3844 ; 2198-3844
ISSN (online)	2198-3844
ISSN	2198-3844
DOI	10.1002/advs.202302229
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Article ; Online: Microtubule-Actomyosin Mechanical Cooperation during Contact Guidance Sensing.

Tabdanov, Erdem D / Puram, Vikram / Zhovmer, Alexander / Provenzano, Paolo P

Cell reports

2018 Volume 25, Issue 2, Page(s) 328–338.e5

Abstract: Cancer cell migration through and away from tumors is driven in part by migration along aligned extracellular matrix, a process known as contact guidance (CG). To concurrently study the influence of architectural and mechanical regulators of CG sensing, ... ...

Abstract	Cancer cell migration through and away from tumors is driven in part by migration along aligned extracellular matrix, a process known as contact guidance (CG). To concurrently study the influence of architectural and mechanical regulators of CG sensing, we developed a set of CG platforms. Using flat and nanotextured substrates with variable architectures and stiffness, we show that CG sensing is regulated by substrate stiffness and define a mechanical role for microtubules and actomyosin-microtubule interactions during CG sensing. Furthermore, we show that Arp2/3-dependent lamellipodia dynamics can compete with aligned protrusions to diminish the CG response and define Arp2/3- and Formins-dependent actin architectures that regulate microtubule-dependent protrusions, which promote the CG response. Thus, our work represents a comprehensive examination of the physical mechanisms influencing CG sensing.
MeSH term(s)	Actin Cytoskeleton/metabolism ; Actomyosin/metabolism ; Breast Neoplasms/physiopathology ; Cell Adhesion ; Cell Communication ; Cell Movement ; Extracellular Matrix/metabolism ; Female ; Humans ; Microtubules/metabolism ; Pseudopodia/physiology ; Tumor Cells, Cultured
Chemical Substances	Actomyosin (9013-26-7)
Language	English
Publishing date	2018-10-10
Publishing country	United States
Document type	Journal Article ; Research Support, N.I.H., Extramural ; Research Support, Non-U.S. Gov't
ZDB-ID	2649101-1
ISSN	2211-1247 ; 2211-1247
ISSN (online)	2211-1247
ISSN	2211-1247
DOI	10.1016/j.celrep.2018.09.030
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Article ; Online: Engineering Elastic Nano- and Micro-Patterns and Textures for Directed Cell Motility

Erdem D. Tabdanov / Alexander S. Zhovmer / Vikram Puram / Paolo P. Provenzano

STAR Protocols, Vol 1, Iss 1, Pp 100013- (2020)

2020

Abstract: Summary: We present a reproducible protocol for fabrication of polyacrylamide (PAA) hydrogel-based nano-patterns and nano-textures with a wide range of elastic rigidities to study fundamental cell behaviors, such as mechanosensitivity and motility. We ... ...

Abstract	Summary: We present a reproducible protocol for fabrication of polyacrylamide (PAA) hydrogel-based nano-patterns and nano-textures with a wide range of elastic rigidities to study fundamental cell behaviors, such as mechanosensitivity and motility. We explore the benefits of this protocol by successfully testing the compatibility of the PAA platforms with super-resolution microscopy, which is largely unavailable with platforms of nano-scale textures made from different polymers. We also utilized soft and rigid nano-textures to study the mechanosensing basis of T cell behavior and phenotype.For complete information on the generation and use of this protocol, please refer to Tabdanov et al. (2018b).
Keywords	Science (General) ; Q1-390
Language	English
Publishing date	2020-06-01T00:00:00Z
Publisher	Elsevier
Document type	Article ; Online
Database	BASE - Bielefeld Academic Search Engine (life sciences selection)

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