August 21, 2014

Microtubules are known for their fascinating dynamics, but some cellular processes require a more stable microtubule cytoskeleton. Thankfully, these stable, acetylated microtubules are just as photogenic as their non-modified microtubule pals. Today’s image is from a paper describing the role of the protein paxillin in microtubule acetylation. 

Crawling cells require coordination of adhesive forces, cytoskeletal rearrangements, and cell polarization. Cell polarization helps to direct newly synthesized proteins to the leading edge of the crawling cell, relying on both a stable microtubule cytoskeleton and positioning of the Golgi apparatus in front of the nucleus. The stability of these long-lived microtubules is due to acetylation—a post-translational modification of α-tubulin. A recent study by Deakin and Turner uncovered a role for the focal adhesion scaffolding protein paxillin in regulating microtubule acetylation, which in turn regulates Golgi integrity and cell polarization. Paxillin modulates microtubule stability through its inhibition of HDAC6, an α-tubulin deacetylase, and does so in both normal and transformed cells. In the images above, depletion of paxillin (bottom) in malignant (left column) and normal (right column) cell types resulted in a drop of microtubule acetylation (yellow), compared to control cells (top).

Deakin, N., & Turner, C. (2014). Paxillin inhibits HDAC6 to regulate microtubule acetylation, Golgi structure, and polarized migration originally published in the Journal of Cell Biology, 206 (3), 395-413 DOI: 10.1083/jcb.201403039

July 10, 2013

The grace of a migrating cell is as deceiving as a pair of Spanx on an English Bulldog… there is a lot going underneath.  Today’s image is from a paper showing the importance of the protein vinculin at the leading edge of a migrating cell.

Cell migration is driven by actin filament polymerization that pushes the leading edge of the cell forward, as well as F-actin retrograde flow.  Focal adhesions (FAs) adhere the crawling cell to the underlying extracellular matrix (ECM), and are assembled and disassembled near the leading edge of the cell.  Proteins of these FAs are believed to make up a “molecular clutch” that engages the retrograde F-actin flow, and a recent paper identifies the protein vinculin, an actin-binding protein, as a molecular clutch.  Thievessen and colleagues investigated the effects of vinculin gene disruption in migrating fibroblasts, and found that vinculin is important in regulating F-actin organization and FA dynamics.  Specifically, vinculin generates the ECM traction forces necessary for migration, and promotes FA formation and turnover.  In the images above, a normal fibroblast (top) and a fibroblast lacking vinculin (bottom) show F-actin (green) and the lamellipodial protein cortactin (purple).  Normal crawling fibroblasts have a sharply defined band of cortactin colocalized with F-actin at the leading edge, while vinculin mutants have a wider, less defined region of cortactin at the leading edge, suggesting the importance of vinculin in leading edge organization.

BONUS!  Check out some cool movies from this paper here.

Thievessen I, Thompson PM, Berlemont S, Plevock KM, Plotnikov SV, Zemljic-Harpf A, Ross RS, Davidson MW, Danuser G, Campbell SL, & Waterman CM (2013). Vinculin-actin interaction couples actin retrograde flow to focal adhesions, but is dispensable for focal adhesion growth. originally published in the Journal of Cell Biology, 202 (1), 163-77 PMID: 23836933

January 7, 2013

Happy New Year! To kick off 2013 properly, today I’m featuring an image from a lab whose leader is one of the coolest cats in cell biology (click here for proof). The image is from a paper describing the nanoscale forces within individual focal adhesions, and how these forces guide cell migration.

“Durotaxis” is the directed cell migration along a rigidity gradient—meaning the migration toward a more mechanically stiff extracellular matrix (ECM). Durotaxis occurs throughout development and cancer metastasis, two situations during which the stiffness of a tissue can vary locally or over time. A recent paper describes how cells sample the environment to sense ECM stiffness, and how this sampling guides migration of a cell toward stiff ECM. Using high-resolution time-lapse traction force microscopy, Plotnikov and colleagues documented the forces within individual focal adhesions (FAs) in migrating cells. These individual FAs tug on the ECM to function as local sensors of ECM rigidity. This tugging traction is necessary for durotaxis, but not chemotaxis, another type of cell migration. In the images above, paxillin is fluorescently labeled to show FAs (top, left and zoomed image on right). The corresponding heatmaps (bottom, left and right) show the magnitudes of tugging forces on the ECM (FAs outlined in black), while the stress vector field image (middle right) shows the direction of the forces within the FAs.

Plotnikov, S., Pasapera, A., Sabass, B., & Waterman, C. (2012). Force Fluctuations within Focal Adhesions Mediate ECM-Rigidity Sensing to Guide Directed Cell Migration Cell, 151 (7), 1513-1527 DOI: 10.1016/j.cell.2012.11.034

Copyright ©2013 Elsevier Ltd. All rights reserved. 

October 16, 2012

Molecular switches are an elegant way for a cell to regulate countless processes with a limited number of proteins. The layers of regulation that drive switch activation or inactivation allow the cell to drive one very specific process, despite the presence of all of the tools and materials to drive a thousand other processes. Today’s image is from a paper that describes one layer of regulation on a well-studied (and well-loved) set of molecular switches.

Rho GTPases are actin regulators that switch between active and inactive states. Their activation state is regulated by proteins (GEFs) that drive the GTPases into their active state, and others (GAPs) that drive them into their inactive state. RhoGDIs (Rho guanine nucleotide dissociation inhibitors) provide an additional level of control over Rho GTPase activity by sequestering Rho GTPases in inactive complexes. The selective dissociation of individual Rho GTPases from these complexes provides the cell with a context-specific response based on the actin-based structures required. A recent paper describes the function of a protein called diacylglycerol kinase ζ (DGKζ) in the release of two Rho GTPases, Rac1 and RhoA, from RhoGDI complexes. Rac1 regulates membrane ruffling and lamellipodia formation, while RhoA regulates stress fiber and focal adhesion formation. Ard and colleagues found that DGKζ-deficient cells showed signs of faulty RhoA signaling, as seen in the images above. Actin stress fibers (left column, green in merged) and focal adhesions (middle column, red in merged) appear normal in wild-type cells (top). In DGKζ-null cells (bottom row), actin stress fibers appeared condensed (arrow) and less organized, while focal adhesion distribution was impaired.

Ard, R., Mulatz, K., Abramovici, H., Maillet, J., Fottinger, A., Foley, T., Byham, M., Iqbal, T., Yoneda, A., Couchman, J., Parks, R., & Gee, S. (2012). Diacylglycerol kinase regulates RhoA activation via a kinase-independent scaffolding mechanism Molecular Biology of the Cell, 23 (20), 4008-4019 DOI: 10.1091/mbc.E12-01-0026

February 10, 2011

There are times when you finish reading a paper and you push it in front of your labmates and ask, “Did you seeeeee this?!” Hopefully, your labmates will indulge you as you do an interpretive dance explaining the coolness of the paper. If I were still in a lab, the images and paper in today’s post would be thrust in front of my labmates….great results and killer microscopy!

Focal adhesions are cellular structures that connect a cell to the underlying extracellular matrix, and play important roles in cell migration, cytoskeletal regulation, and signaling. The complex protein composition and dynamics within focal adhesions have made it difficult to understand their precise architecture. A recent paper uses a new technique called iPALM to determine the nanoscale organization of focal adhesions. This technique allowed for super-resolution images of focal adhesion components, with the added bonus of data indicating the exact depth of each component. From this data, Kanchanawong and colleagues were able to build a precise three-dimensional model of focal adhesion organization. Images above show the localization of integrin and actin using iPALM, with color-coding indicating the depth of each protein relative to the plasma membrane of the cell. When looking at a side view of the focal adhesion (bottom images), the yellow coding of integrin reveals that it resides closely to the plasma membrane on the coverglass (indicated by 0), while actin was found deeper in the cell.

Adapted by permission from Macmillan Publishers Ltd, copyright 2010.

Kanchanawong, P., Shtengel, G., Pasapera, A., Ramko, E., Davidson, M., Hess, H., & Waterman, C. (2010). Nanoscale architecture of integrin-based cell adhesions Nature, 468 (7323), 580-584 DOI: 10.1038/nature09621