Matrix Compliance

Analyses of cell migration in 3D matrices show that maximal migration speeds are observed in matrices of either lesser stiffness or greater ligand density as integrin receptors are blocked by antibody, depending on two mechanical force balances.

The speed of cells migrating in 2D is governed by traction and adhesion forces. However, unlike migration on a 2D surface, the traction forces exerted by a cell moving in a 3D matrix are transmitted throughout the entire surface of the cell. Therefore, additional parameters, such as matrix stiffness pore size are also likely to influence migration in 3D.

In PNAS, Matsudaira and colleagues examined whether ligand and receptor levels display a similar relationship in 2- and 3D environments in relation to migration velocity. Whereas in 2D environments cells compensate for a decrease in the number of receptors by shifting the maximum speed to higher ligand densities, cells in 3D matrices shift their maximum speed to lower traction forces. The authors propose that the stiffness of the matrigel, which is crucial for cell traction in 3D, could account for this diametrically opposite behavior. Other factors such as pore size and proteolysis were also found to influence migration in 3D. This paper describes a new set of balances between cell and matrix properties that govern migration velocity in 3D. Click here for a Whitehead Institute's press release on this article

1. Zaman MH, Trapani LM, Siemeski A, Mackellar D, Gong H, Kamm RD, Wells A, Lauffenburger DA, Matsudaira P. Migration of tumor cells in 3D matrices is governed by matrix stiffness along with cell-matrix adhesion and proteolysis. Proc Natl Acad Sci U S A. 2006 Jul 18;103(29):10889-94 PubMed

Actin Arginylation

The post-translational addition of an Arg residue to the N terminus of beta-actin prevents actin filaments from aggregating and facilitates lamella formation.

This month in Science Karakozova et al. show for the first time that beta-actin is arginylated in vivo. By analyzing the actin isoforms in whole-cell lysates they established that Arg localizes on the N-terminus of beta-actin's Asp3.

To determine the effects of this modification the authors examined the cytoskeleton of cells from Ate^-/- mice, which lack the enzyme required for arginylation. While the stability of actin filaments was unaffected, random filamentous aggregates were observed. Furthermore, Ate^-/- cells were unable to form normal lamella and exhibited defective spreading. Interestingly, transient transfection with a permanently arginylated beta-actin construct restored lamella formation in Ate^-/- cells, confirming that the defects were due to the absence of beta-actin arginylation.

A mathematical model of actin assembly in the presence and absence of arginylation confirmed the experimental data, and supports the idea that 'bulky' Arg residues at the sides of actin filaments decrease the rate of counter-ion-mediated bundling. By preventing filament aggregation, arginylation facilitates the formation of a loose actin network and cell motility.

1. Karakozova M, Kozak M,Wong CC, Bailey AO,Yates JR 3rd, Mogilner A, Zebroski H, Kashina A. Arginylation of Beta Actin Regulates Actin Cytoskeleton and Cell Motility. Science. 2006 Jul 14;313(5784):192-6. PubMed

Other Publications

1. Karakozova M, Kozak M, Wong CC, Bailey AO, Yates Iii JR, Mogilner A, Zebroski H, Kashina A. Arginylation of Beta Actin Regulates Actin Cytoskeleton and Cell Motility. Science. 2006 Jun 22; [Epub ahead of print] PubMed

2. Moissoglu K, Slepchenko BM, Meller N, Horwitz AF, Schwartz MA. In vivo dynamics of Rac-membrane interactions. Mol Biol Cell. 2006 Jun;17(6):2770-9. PubMed

3. Webb DJ, Kovalenko M, Whitmore L, Horwitz AF. Phosphorylation of serine 709 in GIT1 regulates protrusive activity in cells. Biochem Biophys Res Commun. 2006 Aug 11;346(4):1284-8. Epub 2006 Jun 14. PubMed

4. Marx J. Cell biology. Podosomes and invadopodia help mobile cells step lively. Science. 2006 Jun 30;312(5782):1868-9. PubMed

Disturbances in the Matrix

EGFR kinase, ERK, m-calpain, Rho kinase and myosin light chain kinase mediate the effect of EGF on matrix deformation.

During wound healing migrating fibroblasts exert mechanical force on the extracellular matrix causing matrix deformations. EGF has been shown to stimulate contractility and promote cell migration but its effects on matrix compaction have not been explored. The authors show that EGF induces a dose-dependent compaction of collagen matrices and shed light on some of the signaling components involved.

In vitro fibroblast-embedded collagen matrix compaction assays reveal that EGF induces a biphasic response, peaking at 10 ng/ml. Cells expressing kinase dead EGFR were unable to induce matrix deformations upon stimulation with optimal concentrations of EGF. Similarly, treatment with a MEK inhibitor prevented EGF-induced matrix compaction, suggesting that signaling through the EGF receptor and the ERK/MAPK pathway is required for this effect. Interestingly intermediate concentrations of the calpain inhibitor ALLN enhances compaction, but high ALLN concentrations or siRNA mediated downregulation of M-calpain, an ERK effector which stimulates cell motility by allowing the release of the cell's rear, diminish EGF-induced compaction. These results suggest that calpain could be an important regulator of contractile force transmission to the matrix and thus, wound repair. Finally, the authors also show that EGF-induced matrix compaction also involves MLCK and Rho Kinase further supporting the idea that matrix compaction involves multiple signaling pathways ractile forces and adhesiveness.

1. Smith KD, Wells A, Lauffenburger DA. Multiple signaling pathways mediate compaction of collagen matrices by EGF-stimulated fibroblasts. Exp Cell Res. 2006 Apr 2; [Epub ahead of print] PubMed

Myosin V's structure

The three-dimensional structure of Myosin V's inactive state sheds new light on how the motor is regulated.

Myosin V is an actin based motor responsible for short range transport. In neuronal cells, it is important for bringing vesicles to the plasma membrane and sustaining lamellar protrusion. The Taylor laboratory has employed cryoelectron microscopy to elucidate the structure of Myosin V in the inactive state. Their findings, described in Nature, show that unlike Myosin II, the inactive state of Myosin V strongly binds actin, which has important implications for motor recycling. The authors suggest that once cargo is delivered (to the actin +end), rather than being transported back to its starting point by another motor protein or simple diffusion, actin treadmilling ensures that the motor reaches the -end of the filament where it can bind new cargo. The data also indicate that the reduced ATPase activity of the inactive state could be due to a decrease in the rate of nucleotide exchange. This study not only provides new insights into how Myosin V works, but also highlights differences between the regulatory mechanisms of distinct actin motor proteins.

1. Liu J, Taylor DW, Krementsova EB, Trybus KM, Taylor KA. Three-dimensional structure of the myosin V inhibited state by cryoelectron tomography. Nature advance online publication 16 April 2006. PubMed

Proteomic data mining

The data mining tool BlastPro reveals alpha-actinin as a biomarker of metastatic potential

The Klemke laboratory has developed BlastPro, a powerful new data mining tool that is fully automated and facilitates comparison of large proteomic and genomic datasets across species, databases and platforms. BlastPro has numerous applications, including screening for disease-related biomarkers, finding perturbed gene expression patterns in response to environmental cues, or comparison and validation of phosphoprotein sites. Using BlastPro, Klemke and colleagues analyzed data from 5 independent, genomic and proteomic studies comprised of more than 106 genomic sequences and more than 1000 proteins. The cytoskeletal protein alpha-actinin was found to be strongly amplified in metastatic breast, prostate and skin cancers, and is highly enriched in the pseudopodia of invasive cells. These results indicate that alpha-actinin could serve as a biomarker of metastatic potential and demonstrate how BlastPro can broaden the impact of genomic and proteomic studies. BlastPro is freely available on the Cell Migration Consortium website (see New postings below).

1. Wang Y, Hanley R, Klemke RL. Computational Methods for Comparison of Large Genomic and Proteomic Datasets Reveal Protein Markers of Metastatic Cancer. J Proteome Res. 2006 Apr;5(4) :907-15. PubMed

Imaging

New fluorescent biosensor to visualize spatiotemporal dynamics of RhoA activity during cell migration.

Typically RhoA is thought to regulate contractility at the rear end of the cell, but now, using a biosensor with intramolecular fluorescence resonance energy transfer (FRET) that responds to RhoA activation, Hahn et al. demonstrate a role for RhoA activity in membrane protrusion. In a study published in Nature, they show that in randomly migrating mouse embryonic fibroblast cells (MEFs) a sharp band of RhoA activity localizes to the leading edge, while only sporadic bursts of activity were observed in the retracting tails. In contrast, during directed migration (platelet derived growth factor (PDGF)-induced), low levels of RhoA activity were observed at the leading edge. The authors confirm that this effect is due to the PDGF-induced activation of Rac, which leads to a downregulation of RhoA activity. This study shows for the first time that the localization of RhoA activity at the leading edge is different in cells undergoing directed migration compared to those that are randomly migrating.

1. Pertz O, Hodgson L, Klemke RL, Hahn KM. Spatiotemporal dynamics of RhoA activity in migrating cells. Nature, 2006 Mar 19; [Epub ahead of print] PubMed

Breast Cancer 1000 library

A collection of cDNAs are generated that are implicated in breast carcinogenesis - the Breast Cancer 1000 (BC1000)

BC1000 is the first publicly available sequence-validated human disease gene collection. It contains over 1,300 breast cancer-related genes, although the actual role many of them have in tumorigenesis is yet to be determined. It is hoped that BC1000 will enable the systematic investigation of how these genes function and accelerate the development of therapeutic agents.

A study published in the Journal of Proteome Research, describes an investigation of the functional activity of 265 genes from the BC1000 collection. The authors examined changes in proliferation, migration and morphogenesis in immortalized but benign breast epithelial cells (MCF-10A cells) expressing these genes. The collection includes genes such as IL4, IL11 and IL13 which stimulate migration of immortalized breast epithelial cells. Similarly, SGK (serum and glucocorticoid-regulated kinase-1) and TNFRSF10B (tumor necrosis factor receptor, 10B) also seem to have a pro-migratory role, despite previously being recognized for their role in cell survival. Interestingly, only 9% of the genes examined gave positive results in the proliferation assay, whereas 22% and 29% gave positive results for the morphogenesis assay and the migration assay, respectively. These findings highlight the importance of genes regulating cell invasiveness during breast carcinogenesis.

1. Witt AE, Hines LM, Collins NL, Hu Y, Gunawardane RN, Moreira D, Raphael J, Jepson D, Koundinya M, Rolfs A, Taron B, Isakoff SJ, Brugge JS, Labaer J. Functional Proteomics Approach to Investigate the Biological Activities of cDNAs Implicated in Breast Cancer. J Proteome Res. 2006 Mar;5(3):599-610. PubMed

Data postings to the CMC Activity Center

Preliminary data from ASCB and Biophysical Society poster presentations

Brugge & Jacobson laboratory posters from ASCB & Biophysical Society meetings are posted on the Discovery - Genetics/Screen, www.cellmigration.org/resource/discovery/ discov_activities.shtml#genes imaging, Imaging & Photomanipulation www.cellmigration.org/resource/imaging/ and Modeling initiative www.cellmigration.org/resource/modeling/activity pages in the CMC Activity Center.

C. elegans gonad formation - Primary RNAi screen results

Feeding-mediated RNAi of C. elegans was used to screen for genes that are required for normal gonad formation and distal tip cell migration. The table presented via the Discovery -Genetics/Screens activity table www.cellmigration.org/resource/discovery/discov_activities.shtml gives the GenePairs ID for each gene that scored positive in the primary screen. Each of these genes was further tested by DIC microscopy in the secondary RNAi screen. Gonad defects were confirmed for about 20% of these genes.

Data on new phosphorylation sites posted

Phosphorylation sites on several key migration related proteins have recently been added to the Discovery - Proteomics page in the CMC Activity Center ...www.cellmigration.org/resource/discovery/

New binding partners data posted

Binding partner data for several key migration related proteins have recently been added to the Discovery - Proteomics page in the CMC Activity Center... www.cellmigration.org/resource/discovery/#binding

Other Publications

Prostate derived Ets factor (PDEF) involved in breast tumorigenesis

The Brugge laboratory has identified factors that stimulate cell migration and there by contribute to oncogenic progression. Using an MCF7 cDNA library and the immortalized human breast epithelial cell line MCF-10A they conducted a powerful retroviral based migration screen and identified prostate derived Ets factor (PDEF) as a candidate promigratory gene. Alone PDEF has little effect on MCF-10A cell migration, but when expressed with receptor tyrosine kinases ErbB2 and colony-stimulating factor receptor (CSF-1R)/CSF-1 it produced a dramatic invasive phenotype (Gunawardane et al., 2005).

1. Gunawardane RN, Sgroi DC, Wrobel CN, Koh E, Daley GQ, Brugge JS. Novel role for PDEF in epithelial cell migration and invasion. Cancer Res. 2005 Dec 15;65 (24):11572-80. PubMed

The level set method as a numerical scheme to reconstruct continuous boundary movement in time-lapse image sequences with finite time sampling

The Danuser group propose a framework for tracking arbitrary complex cell boundary movements, relying on a unique definition of protrusion and retraction as the pathlength a virtual edge marker traverses when moving continuously perpendicular to the cell boundary. They introduce the level set method as a numerical scheme to reconstruct continuous boundary movement in time-lapse image sequences with finite time sampling. For moderately complex movements, they describe a numerically less expensive method that satisfactorily approximates the definition. Densely sampled protrusion and retraction rates were accumulated in space-time charts revealing distinct morphodynamic states. Their data support a model where activation of Rac1 mediates the propagation of protrusion waves, whose persistence depends on the relative abundance of activated Arp2/3 and polymerizable G-actin.

1. Machacek M, Danuser G. Morphodynamic profiling of protrusion phenotypes. Biophys J. 2006 Feb;90(4):1439-52. PubMed

Electron microscopy

Electron microscopy and computational analysis reveal the structural basis of actin filament nucleation and branch stability

The Arp2/3 complex is crucial for generating branched actin filament networks at the leading edge of motile cells. This evolutionarily conserved 220 kDa complex consists of seven subunits that include two actin-related proteins (Arps), Arp2 and Arp3, and five additional subunits named ARPC1 to ARPC5. Assembly of actin branch junctions requires the Arp2/3 complex to initiate a new actin (daughter) filament branch from the side of an existing (mother) filament. Using genetic labelling and electron microscopy, Egile and colleagues have determined the structural organization of actin branch junctions assembled in vitro with 1 nm precision. They show that the activated Arp2/3 complex binds to the side of the pre-existing (mother) filaments through multiple complex subunits. These findings provide direct structural insights into the mechanism of branch junction assembly, in which Arp2 and Arp3 form a template at the branch junction that triggers daughter filament growth.

1. Egile C, Rouiller I, Xu XP, Volkmann N, Li R, Hanein D. Mechanism of filament nucleation and branch stability revealed by the structure of the Arp2/3 complex at acting branch junctions. PLoS Biol. 2005 Nov;3(11):e383. PubMed

Structural Biology

A CryoEM study reveals the structural basis of Myosin V's processive movement

In a recent cryoEM study of myosin V bound to actin, Volkmann and colleagues were able to visualize the previously inaccessible 'weakly-bound' states of the actomyosin ATPase cycle thanks to myosin V's higher affinity for actin in the presence of ATP and nucleotide analogues. Electron cryomicroscopy together with computer-based docking of crystal structures into three-dimensional reconstructions, were used to create atomic models of actin-bound myosin V in the strong-binding states (in the presence of ADP and in the absence of nucleotide), and in the weak-binding states (in the presence of ATP and AMPPNP/AlF4). Like in myosin II, loop 1, 2 and the actin-binding cleft of myosin V are key structural elements participating in the actin-myosin interaction. In this study, they expanded their analysis and determined that loop 2 serves as a tether that allows myosin V to remain attached to actin during the hydrolysis cycle. They also show that the actin-binding cleft opens in the presence of ATP, and that it can significantly change the actomyosin interface in the case of myosin V. This ATP-triggered cleft opening is most likely isoform independent and provides a mechanism for dissociation from actin in the case of myosin II. Finally, the authors provide the first direct visual evidence that the actin-attached pre-power stroke conformation of the lever arm correlates with the transition state of the myosin hydrolysis cycle (ADP.AlF4).

1. Volkmann N, Liu H, Hazelwood L, Krementsova EB, Lowey S, Trybus KM, Hanein D. The structural basis of Myosin V processive movement as revealed by electron cryomicroscopy. Mol Cell. 2005 Sep 2;19(5):595-605. PubMed

New software download available

GLOBALS software and tutorials for image analysis are available for correlation microscopy

The GLOBALS software can be downloaded for a free 30 day trial period by visiting: www.cellmigration.org/resource/imaging/imaging_resources.shtml#protocols. These are new programmes for fluorescence correlation spectroscopy (FCS) using images accumulated by laser scanning confocal microscopy. The software implements raster image correlation spectroscopy (RICS) analyses (Digman et al., 2005 a&b), and fluorescence lifetime images using the phasor approach. These programmes bring a powerful temporal capability to inexpensive, standard, calibrated laser scanning confocal microscopes (LSCM). Tutorials on these programs and their utility can also be downloaded as powerpoint presentations.

1. Digman MA, Brown CM, Sengupta P, Wiseman PW, Horwitz AR, Gratton E. Measuring fast dynamics in solutions and cells with a laser scanning microscope. Biophys J. 2005a Aug;89(2):1317-27. PubMed

2. Digman MA, Sengupta P, Wiseman PW, Brown CM, Horwitz AR, Gratton E. Fluctuation correlation spectroscopy with a laser-scanning microscope: exploiting the hidden time structure. Biophys J. 2005b May;88(5):L33-6. PubMed

Chemotaxis

A new study highlights the simplicity of gradient sensing in fibroblasts

Using a novel combination of mathematical modelling and experimentation, Schneider and Haugh have discovered that fibroblasts apparently lack the sophisticated signaling loops that are used in neutrophils to sense gradients; instead, they need more strict instructions. Neutrophils are able to amplify small differences in chemoattractant levels at the front and back of the cell and convert them into large differences in phosphatidyl inositol (PI) production that polarize cytoskeletal dynamics. Furthermore, they are able to adapt quickly to uniform chemoattractant levels by returning PI to basal levels. PDGF-stimulated fibroblasts, however, are unable to adapt in this way. To understand how a fibroblast’s sensing mechanism works, Schneider and Haugh formulated a mathematical model to describe the simplest possible mechanism: PDGF-bound receptors activate PI3K locally, leading to the production of PI. Receptors would then compete for limiting amounts of PI3K, without global positive feedback loops. The model correctly predicts the PDGF gradient-sensing behavior of fibroblasts under all tested conditions. This simplicity comes at a price: fibroblasts have a narrower range (by an order of magnitude) of chemoattractant concentrations within which they will respond well to, compared to neutrophils. They cannot, therefore, sense shallow gradients. It is tempting to speculate that this simplicity evolved because, unlike neutrophils, fibroblasts have to sense long lasting and steep PDGF gradients in tissue wounds.

1. Schneider IC, Haugh JM. Quantitative elucidation of a distinct spatial gradient-sensing mechanism in fibroblasts. J Cell Biol. 2005 Dec 5;171(5):883-92. Epub 2005 Nov 28. PubMed

Mathematics at the leading edge

State-of-the-art mathematical models of protrusion are reviewed for the first time

Actin-based protrusion is the first step in cell crawling. In the past two decades, the studies of actin networks in the lamellipodium and of Listeria's comet tail movement have advanced to the extent that the last goal of the reductionist agenda - reconstitution of protrusion from purified components in vitro and in silico - has become viable. Earlier models dealt with growth of, and force generation by, a single actin filament. Recent models of tethered ratchet, autocatalytic branching, end-tracking motor action and elastic- and nano- propulsion have helped to elucidate the dynamics and forces operating in complex actin networks. By considering these models, their limitations and their relationships to recent biophysical data, Alex Mogilner discusses the progress being made towards a unified model of protrusion.

1. Mogilner A. On the edge: modeling protrusion. Curr Opin Cell Biol. 2005 Nov 26; [Epub ahead of print] PubMed

Lamellipodium vs lamella

Whilst the lamellipodium steers the cell in the right direction, the lamella is the engine that drives cell movement

Until now, the lamellipodium was considered to be the engine driving cell motility at the leading edge of the cell. Surprisingly, a large interdisciplinary team of researchers that includes Gaudenz Danuser and Dorit Hanein, have discovered that lamellipodia can be disrupted without stopping cell advance. In fact, cells moved faster without lamellipodia. The actual cell-driving device appears to be the lamella, the structure that lies behind the lamellipodium. To show this, the team injected tropomyosin, which normally resides in lamellae and is excluded from lamellipodia, into the cells. This disrupted important lamellipodial molecules, including Arp2/3 and ADF/cofilin, and actin treadmilling, but did not block retrograde actin flow in the lamella. Surprisingly, cells with elevated tropomyosin doubled their migration speed. This study provocatively hypothesizes that lamellipodia are involved in the rapid responses to extracellular cues, while lamellae are the actual engines driving movement.

1. Gupton SL, Anderson KL, Kole TP, Fischer RS, Ponti A, Hitchcock-DeGregori SE, Danuser G, Fowler VM, Wirtz D, Hanein D, Waterman-Storer CM. Cell migration without a lamellipodium: translation of actin dynamics into cell movement mediated by tropomyosin. J Cell Biol. 2005 Feb 14;168(4):619-31. PubMed