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Related Concept Videos

Small GTPases - Ras and Rho01:24

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Ras and Rho are small monomeric GTPases that act downstream of receptor tyrosine kinase (RTK) and regulate various cellular processes. These GTPases switch between active and inactive states by binding to guanine nucleotides.
Three regulatory proteins control their activity:
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Activation and Inactivation of G Proteins01:22

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Heterotrimeric G proteins are guanine nucleotide-binding proteins. As the name suggests, heterotrimeric G proteins are composed of three subunits: alpha, beta, and gamma. They remain GDP-bound or GTP-bound inside the cells and switch between inactive/active states. The Gα subunit possesses the nucleotide-binding pocket that binds guanine nucleotides and switches between GDP or GTP-bound states. In contrast, the Gꞵ and Gγ subunits are always bound together with high...
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Osteoclasts are cells responsible for bone resorption and remodeling. They originate from hematopoietic progenitor cells present in the bone marrow. Numerous progenitor cells fuse to form multinucleated cells, each with 10-20 nuclei. A single osteoclast has a diameter of 150 to 200 µM. These cells have ruffled borders that break down the underlying bone tissue and release minerals such as calcium into the blood in bone resorption. Osteoclasts cling to bones with their ruffled edges during...
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Cell Polarization by Rho Proteins01:21

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Cell polarity is the asymmetric distribution of cellular and membrane components, making one side of the cell different from the other. This polarity is essential to many processes such as embryogenesis, axon migration, glucose transport across epithelial cells, and directional cell migration. A migrating cell responds to intracellular or extracellular signals via molecular cascades that reorganize the actin cytoskeleton to establish this polarity. In these cells, the Rho family proteins Cdc42,...
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Guanine nucleotide-binding proteins (G-proteins), also known as GTPases, are a superfamily of proteins that regulate many cellular processes, such as cell signaling, vesicular transport, and the regulation of cell shape and motility. Mutation or dysfunction of these proteins can lead to disease. There are around 40,000 known G-proteins that can broadly be classified into two groups ‒  small G-proteins consisting of a single domain and large multi-domain G-proteins.
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Rab GTPases act in a regulated cascade during membrane fusion, helping the lipid bilayers mix. The Rab family of proteins are active when bound to GTP, and inactive when bound to GDP. Hence, they act as guanine nucleotide-dependent molecular switches. Rab-GTP recognizes and binds to long or short-range tethering proteins to capture the target vesicle. These tethers coordinate with SNAREs on the vesicle and the target membrane to assemble the trans SNARE complex that locks the mixing bilayers.
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Related Experiment Video

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Isolation, Purification, and Differentiation of Osteoclast Precursors from Rat Bone Marrow
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Rho GTPase techniques in osteoclastogenesis.

Roland Leung1, Michael Glogauer

  • 1Matrix Dynamics Group, University of Toronto, Toronto, ON, Canada.

Methods in Molecular Biology (Clifton, N.J.)
|December 7, 2011
PubMed
Summary

This study simplifies in vitro osteoclast culturing and examines Rho GTPases, key regulators of the actin cytoskeleton, which is vital for osteoclast function, adhesion, and migration.

Area of Science:

  • Cell Biology
  • Biochemistry
  • Biotechnology

Background:

  • Osteoclast in vitro culturing historically required co-culturing monocytes with bone marrow stromal cells.
  • Receptor activator of nuclear factor kappa B ligand (RANKL) has simplified osteoclast culturing.
  • The actin cytoskeleton is crucial for osteoclast structural support, adhesion, polarization, and migration.

Purpose of the Study:

  • To describe simple techniques for culturing primary osteoclasts from murine bone marrow cells.
  • To evaluate Rho GTPase activation states in osteoclasts.
  • To measure monocyte migratory abilities and introduce proteins into osteoclasts.

Main Methods:

  • Murine bone marrow cell culture for primary osteoclasts.
  • Assessment of Rho GTPase activation states.

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  • Migration assays for monocytes.
  • Protein introduction into osteoclasts via TAT construct.

    • Main Results:

    • Established a simplified method for primary osteoclast in vitro culture.
    • Provided techniques for evaluating Rho GTPase activity in osteoclasts.
    • Developed methods for assessing monocyte migration and protein delivery.

    Conclusions:

    • Simplified osteoclast culturing protocols are presented.
    • Methods for studying Rho GTPase regulation and osteoclast function are detailed.
    • The techniques facilitate research into osteoclast biology and potential therapeutic interventions.