Publications by authors named "Gaudenz Danuser"

136 Publications

Pre-complexation of talin and vinculin without tension is required for efficient nascent adhesion maturation.

Elife 2021 Mar 30;10. Epub 2021 Mar 30.

Lyda Hill Department of Bioinformatics, University of Texas Southwestern Medical Center, Dallas, United States.

Talin and vinculin are mechanosensitive proteins that are recruited early to integrin-based nascent adhesions (NAs). In two epithelial cell systems with well-delineated NA formation, we find these molecules concurrently recruited to the subclass of NAs maturing to focal adhesions. After the initial recruitment under minimal load, vinculin accumulates in maturing NAs at a ~ fivefold higher rate than in non-maturing NAs, and is accompanied by a faster traction force increase. We identify the R8 domain in talin, which exposes a vinculin-binding-site (VBS) in the absence of load, as required for NA maturation. Disruption of R8 domain function reduces load-free vinculin binding to talin, and reduces the rate of additional vinculin recruitment. Taken together, these data show that the concurrent recruitment of talin and vinculin prior to mechanical engagement with integrins is essential for the traction-mediated unfolding of talin, exposure of additional VBSs, further recruitment of vinculin, and ultimately, NA maturation.
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http://dx.doi.org/10.7554/eLife.66151DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8009661PMC
March 2021

SH3BP4 promotes neuropilin-1 and α5-integrin endocytosis and is inhibited by Akt.

Dev Cell 2021 Mar 20. Epub 2021 Mar 20.

Lyda Hill Department of Bioinformatics, UT Southwestern Medical Center, Dallas, TX 75390, USA; Department of Cell Biology, UT Southwestern Medical Center, Dallas, TX, USA. Electronic address:

Cells probe their surrounding matrix for attachment sites via integrins that are internalized by endocytosis. We find that SH3BP4 regulates integrin surface expression in a signaling-dependent manner via clathrin-coated pits (CCPs). Dephosphorylated SH3BP4 at S246 is efficiently recruited to CCPs, while upon Akt phosphorylation, SH3BP4 is sequestered by 14-3-3 adaptors and excluded from CCPs. In the absence of Akt activity, SH3BP4 binds GIPC1 and targets neuropilin-1 and α5/β1-integrin for endocytosis, leading to inhibition of cell spreading. Similarly, chemorepellent semaphorin-3a binds neuropilin-1 to activate PTEN, which antagonizes Akt and thus recruits SH3BP4 to CCPs to internalize both receptors and induce cell contraction. In PTEN mutant non-small cell lung cancer cells with high Akt activity, expression of non-phosphorylatable active SH3BP4-S246A restores semaphorin-3a induced cell contraction. Thus, SH3BP4 links Akt signaling to endocytosis of NRP1 and α5/β1-integrins to modulate cell-matrix interactions in response to intrinsic and extrinsic cues.
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http://dx.doi.org/10.1016/j.devcel.2021.03.009DOI Listing
March 2021

Estimation of the fraction of COVID-19 infected people in U.S. states and countries worldwide.

PLoS One 2021 8;16(2):e0246772. Epub 2021 Feb 8.

Lyda Hill Department of Bioinformatics, University of Texas Southwestern Medical Center, Dallas, Texas, United States of America.

Since the beginning of the coronavirus disease 2019 (COVID-19) pandemic, daily counts of confirmed cases and deaths have been publicly reported in real-time to control the virus spread. However, substantial undocumented infections have obscured the true size of the currently infected population, which is arguably the most critical number for public health policy decisions. We developed a machine learning framework to estimate time courses of actual new COVID-19 cases and current infections in all 50 U.S. states and the 50 most infected countries from reported test results and deaths. Using published epidemiological parameters, our algorithm optimized slowly varying daily ascertainment rates and a time course of currently infected cases each day. Severe under-ascertainment of COVID-19 cases was found to be universal across U.S. states and countries worldwide. In 25 out of the 50 countries, actual cumulative cases were estimated to be 5-20 times greater than the confirmed cases. Our estimates of cumulative incidence were in line with the existing seroprevalence rates in 46 U.S. states. Our framework projected for countries like Belgium, Brazil, and the U.S. that ~10% of the population has been infected once. In the U.S. states like Louisiana, Georgia, and Florida, more than 4% of the population was estimated to be currently infected, as of September 3, 2020, while in New York this fraction is 0.12%. The estimation of the actual fraction of currently infected people is crucial for any definition of public health policies, which up to this point may have been misguided by the reliance on confirmed cases.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0246772PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7869996PMC
February 2021

Imaging assay to probe the role of telomere length shortening on telomere-gene interactions in single cells.

Chromosoma 2021 Mar 8;130(1):61-73. Epub 2021 Feb 8.

Lyda Hill Department of Bioinformatics, UT Southwestern Medical Center, Dallas, TX, USA.

Telomeres are repetitive non-coding nucleotide sequences (TTAGGGn) capping the ends of chromosomes. Progressive telomere shortening with increasing age has been associated with shifts in gene expression through models such as the telomere position effect (TPE), which suggests reduced interference of the telomere with transcriptional activity of increasingly more distant genes. A modification of the TPE model, referred to as Telomere Position Effects over Long Distance (TPE-OLD), explains why some genes 1-10 MB from a telomere are still affected by TPE, but genes closer to the telomere are not. Here, we describe an imaging approach to systematically examine the occurrence of TPE-OLD at the single cell level. Compared to existing methods, the pipeline allows rapid analysis of hundreds to thousands of cells, which is necessary to establish TPE-OLD as an acceptable mechanism of gene expression regulation. We examined two human genes, ISG15 and TERT, for which TPE-OLD has been described before. For both genes, we found less interaction with the telomere on the same chromosome in old cells compared to young cells; and experimentally elongated telomeres in old cells rescued the level of telomere interaction for both genes. However, the dependency of the interactions on the age progression from young to old cells varied. One model for the differences between ISG15 and TERT may relate to the markedly distinct interstitial telomeric sequence arrangement in the two genes. Overall, this provides a strong rationale for the role of telomere length shortening in the regulation of gene expression.
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http://dx.doi.org/10.1007/s00412-020-00747-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7889534PMC
March 2021

Actin-Membrane Release Initiates Cell Protrusions.

Dev Cell 2020 12 11;55(6):723-736.e8. Epub 2020 Dec 11.

Lyda Hill Department of Bioinformatics, University of Texas Southwestern Medical Center, Dallas, TX 75390, USA; Department of Cell Biology, University of Texas Southwestern Medical Center, Dallas, TX 75390, USA. Electronic address:

Despite the well-established role of actin polymerization as a driving mechanism for cell protrusion, upregulated actin polymerization alone does not initiate protrusions. Using a combination of theoretical modeling and quantitative live-cell imaging experiments, we show that local depletion of actin-membrane links is needed for protrusion initiation. Specifically, we show that the actin-membrane linker ezrin is depleted prior to protrusion onset and that perturbation of ezrin's affinity for actin modulates protrusion frequency and efficiency. We also show how actin-membrane release works in concert with actin polymerization, leading to a comprehensive model for actin-driven shape changes. Actin-membrane release plays a similar role in protrusions driven by intracellular pressure. Thus, our findings suggest that protrusion initiation might be governed by a universal regulatory mechanism, whereas the mechanism of force generation determines the shape and expansion properties of the protrusion.
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http://dx.doi.org/10.1016/j.devcel.2020.11.024DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7908823PMC
December 2020

Functional characterization of 67 endocytic accessory proteins using multiparametric quantitative analysis of CCP dynamics.

Proc Natl Acad Sci U S A 2020 12 30;117(50):31591-31602. Epub 2020 Nov 30.

Department of Cell Biology, University of Texas Southwestern Medical Center, Dallas, TX 75390;

Clathrin-mediated endocytosis (CME) begins with the nucleation of clathrin assembly on the plasma membrane, followed by stabilization and growth/maturation of clathrin-coated pits (CCPs) that eventually pinch off and internalize as clathrin-coated vesicles. This highly regulated process involves a myriad of endocytic accessory proteins (EAPs), many of which are multidomain proteins that encode a wide range of biochemical activities. Although domain-specific activities of EAPs have been extensively studied, their precise stage-specific functions have been identified in only a few cases. Using single-guide RNA (sgRNA)/dCas9 and small interfering RNA (siRNA)-mediated protein knockdown, combined with an image-based analysis pipeline, we have determined the phenotypic signature of 67 EAPs throughout the maturation process of CCPs. Based on these data, we show that EAPs can be partitioned into phenotypic clusters, which differentially affect CCP maturation and dynamics. Importantly, these clusters do not correlate with functional modules based on biochemical activities. Furthermore, we discover a critical role for SNARE proteins and their adaptors during early stages of CCP nucleation and stabilization and highlight the importance of GAK throughout CCP maturation that is consistent with GAK's multifunctional domain architecture. Together, these findings provide systematic, mechanistic insights into the plasticity and robustness of CME.
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http://dx.doi.org/10.1073/pnas.2020346117DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7749282PMC
December 2020

Multiplexed GTPase and GEF biosensor imaging enables network connectivity analysis.

Nat Chem Biol 2020 08 18;16(8):826-833. Epub 2020 May 18.

Department of Pharmacology, UNC Chapel Hill, Chapel Hill, NC, USA.

Here we generate fluorescence resonance energy transfer biosensors for guanine exchange factors (GEFs) by inserting a fluorescent protein pair in a structural 'hinge' common to many GEFs. Fluorescent biosensors can map the activation of signaling molecules in space and time, but it has not been possible to quantify how different activation events affect one another or contribute to a specific cell behavior. By imaging the GEF biosensors in the same cells as red-shifted biosensors of Rho GTPases, we can apply partial correlation analysis to parse out the extent to which each GEF contributes to the activation of a specific GTPase in regulating cell movement. Through analysis of spontaneous cell protrusion events, we identify when and where the GEF Asef regulates the GTPases Cdc42 and Rac1 to control cell edge dynamics. This approach exemplifies a powerful means to elucidate the real-time connectivity of signal transduction networks.
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http://dx.doi.org/10.1038/s41589-020-0542-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7388658PMC
August 2020

DASC, a sensitive classifier for measuring discrete early stages in clathrin-mediated endocytosis.

Elife 2020 04 30;9. Epub 2020 Apr 30.

Lyda Hill Department of Bioinformatics, University of Texas Southwestern Medical Center, Dallas, United States.

Clathrin-mediated endocytosis (CME) in mammalian cells is driven by resilient machinery that includes >70 endocytic accessory proteins (EAP). Accordingly, perturbation of individual EAPs often results in minor effects on biochemical measurements of CME, thus providing inconclusive/misleading information regarding EAP function. Live-cell imaging can detect earlier roles of EAPs preceding cargo internalization; however, this approach has been limited because unambiguously distinguishing abortive coats (ACs) from clathrin-coated pits (CCPs) is required but unaccomplished. Here, we develop a thermodynamics-inspired method, "disassembly asymmetry score classification (DASC)", that resolves ACs from CCPs based on single channel fluorescent movies. After extensive verification, we use DASC-resolved ACs and CCPs to quantify CME progression in 11 EAP knockdown conditions. We show that DASC is a sensitive detector of phenotypic variation in CCP dynamics that is uncorrelated to the variation in biochemical measurements of CME. Thus, DASC is an essential tool for uncovering EAP function.
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http://dx.doi.org/10.7554/eLife.53686DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7192580PMC
April 2020

Mechanical regulation of glycolysis via cytoskeleton architecture.

Nature 2020 02 12;578(7796):621-626. Epub 2020 Feb 12.

Lyda Hill Department of Bioinformatics, UT Southwestern Medical Center, Dallas, TX, USA.

The mechanics of the cellular microenvironment continuously modulates cell functions such as growth, survival, apoptosis, differentiation and morphogenesis via cytoskeletal remodelling and actomyosin contractility. Although all of these processes consume energy, it is unknown whether and how cells adapt their metabolic activity to variable mechanical cues. Here we report that the transfer of human bronchial epithelial cells from stiff to soft substrates causes a downregulation of glycolysis via proteasomal degradation of the rate-limiting metabolic enzyme phosphofructokinase (PFK). PFK degradation is triggered by the disassembly of stress fibres, which releases the PFK-targeting E3 ubiquitin ligase tripartite motif (TRIM)-containing protein 21 (TRIM21). Transformed non-small-cell lung cancer cells, which maintain high glycolytic rates regardless of changing environmental mechanics, retain PFK expression by downregulating TRIM21, and by sequestering residual TRIM21 on a stress-fibre subset that is insensitive to substrate stiffness. Our data reveal a mechanism by which glycolysis responds to architectural features of the actomyosin cytoskeleton, thus coupling cell metabolism to the mechanical properties of the surrounding tissue. These processes enable normal cells to tune energy production in variable microenvironments, whereas the resistance of the cytoskeleton in response to mechanical cues enables the persistence of high glycolytic rates in cancer cells despite constant alterations of the tumour tissue.
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http://dx.doi.org/10.1038/s41586-020-1998-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7210009PMC
February 2020

TRA-1-60-positive/CD45 cells found in the peripheral blood of prostate cancer patients with metastatic disease - A proof-of-concept study.

Heliyon 2020 Jan 24;6(1):e03263. Epub 2020 Jan 24.

Genitourinary Oncology, Beth Israel Deaconess Medical Center, Boston, MA, USA.

Purpose: Over 90% of all cancer related deaths are due to metastasis. However, current diagnostic tools can't reliably discriminate between invasive and localized cancers.

Patients And Methods: In this proof-of-concept study, we employed the embryonic stem cell marker TRA-1-60 (TRA+) to identify TRA + cells within the blood of prostate cancer patients and searched for TRA + cells in men with metastatic and localized cancers. We isolated whole peripheral blood mononuclear cells from 26 metastatic prostate cancer patients, from 13 patients with localized prostate cancer and from 17 healthy controls. Cells were stained for DAPI, CD45 and TRA + by immunofluorescence and imaged by epi-fluorescence microscopy. Imaged-based software was used both to identify TRA + cells, and to analyze CD45 levels in TRA+ and negative cells.

Results: We found high numbers of TRA + cells within the blood of metastatic cancer patients, whereas healthy individuals or men with localized prostate cancer showed none or very low numbers of TRA + cells. Further analysis of the CD45 levels of TRA + cells revealed a small population of TRA + cells with almost undetectable CD45 levels that were found frequently in metastatic prostate cancer patients. By excluding CD45 positive cells from the TRA + cell pool, we were able to refine the assay to be highly specific in identifying men with metastatic disease. In fact, the difference of CD45 levels between TRA+ and negative cells was a robust measure to distinguish between men with localized and metastatic prostate cancers in this small patient cohort.

Conclusions: The data suggest that metastatic prostate cancer patient have significant numbers of TRA+/CD45 cells which might represent a potential tool for diagnostic assessment in the future.
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http://dx.doi.org/10.1016/j.heliyon.2020.e03263DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6994489PMC
January 2020

Light-sheet microscopy of cleared tissues with isotropic, subcellular resolution.

Nat Methods 2019 11 31;16(11):1109-1113. Epub 2019 Oct 31.

Department of Cell Biology, University of Texas Southwestern Medical Center, Dallas, TX, USA.

We present cleared-tissue axially swept light-sheet microscopy (ctASLM), which enables isotropic, subcellular resolution imaging with high optical sectioning capability and a large field of view over a broad range of immersion media. ctASLM can image live, expanded, and both aqueous and non-aqueous chemically cleared tissue preparations. Depending on the optical configuration, ctASLM provides up to 260 nm of axial resolution, a three to tenfold improvement over confocal and other reported cleared-tissue light-sheet microscopes. We imaged millimeter-scale cleared tissues with subcellular three-dimensional resolution, which enabled automated detection of multicellular tissue architectures, individual cells, synaptic spines and rare cell-cell interactions.
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http://dx.doi.org/10.1038/s41592-019-0615-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6924633PMC
November 2019

3D flow field estimation and assessment for live cell fluorescence microscopy.

Bioinformatics 2020 03;36(5):1317-1325

Inria, Centre Rennes-Bretagne Atlantique, Rennes 35042, France.

Motivation: The revolution in light sheet microscopy enables the concurrent observation of thousands of dynamic processes, from single molecules to cellular organelles, with high spatiotemporal resolution. However, challenges in the interpretation of multidimensional data requires the fully automatic measurement of those motions to link local processes to cellular functions. This includes the design and the implementation of image processing pipelines able to deal with diverse motion types, and 3D visualization tools adapted to the human visual system.

Results: Here, we describe a new method for 3D motion estimation that addresses the aforementioned issues. We integrate 3D matching and variational approach to handle a diverse range of motion without any prior on the shape of moving objects. We compare different similarity measures to cope with intensity ambiguities and demonstrate the effectiveness of the Census signature for both stages. Additionally, we present two intuitive visualization approaches to adapt complex 3D measures into an interpretable 2D view, and a novel way to assess the quality of flow estimates in absence of ground truth.

Availability And Implementation: https://team.inria.fr/serpico/data/3d-optical-flow-data/.

Supplementary Information: Supplementary data are available at Bioinformatics online.
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http://dx.doi.org/10.1093/bioinformatics/btz780DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7523654PMC
March 2020

Robust and automated detection of subcellular morphological motifs in 3D microscopy images.

Nat Methods 2019 10 9;16(10):1037-1044. Epub 2019 Sep 9.

Lyda Hill Department of Bioinformatics, University of Texas Southwestern Medical Center, Dallas, TX, USA.

Rapid developments in live-cell three-dimensional (3D) microscopy enable imaging of cell morphology and signaling with unprecedented detail. However, tools to systematically measure and visualize the intricate relationships between intracellular signaling, cytoskeletal organization and downstream cell morphological outputs do not exist. Here, we introduce u-shape3D, a computer graphics and machine-learning pipeline to probe molecular mechanisms underlying 3D cell morphogenesis and to test the intriguing possibility that morphogenesis itself affects intracellular signaling. We demonstrate a generic morphological motif detector that automatically finds lamellipodia, filopodia, blebs and other motifs. Combining motif detection with molecular localization, we measure the differential association of PIP and Kras with blebs. Both signals associate with bleb edges, as expected for membrane-localized proteins, but only PIP is enhanced on blebs. This indicates that subcellular signaling processes are differentially modulated by local morphological motifs. Overall, our computational workflow enables the objective, 3D analysis of the coupling of cell shape and signaling.
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http://dx.doi.org/10.1038/s41592-019-0539-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7238333PMC
October 2019

Spatiotemporal dynamics of GEF-H1 activation controlled by microtubule- and Src-mediated pathways.

J Cell Biol 2019 09 16;218(9):3077-3097. Epub 2019 Aug 16.

Deptartment of Bioinformatics, University of Texas Southwestern Medical Center, Dallas, TX

Rho family GTPases are activated with precise spatiotemporal control by guanine nucleotide exchange factors (GEFs). Guanine exchange factor H1 (GEF-H1), a RhoA activator, is thought to act as an integrator of microtubule (MT) and actin dynamics in diverse cell functions. Here we identify a GEF-H1 autoinhibitory sequence and exploit it to produce an activation biosensor to quantitatively probe the relationship between GEF-H1 conformational change, RhoA activity, and edge motion in migrating cells with micrometer- and second-scale resolution. Simultaneous imaging of MT dynamics and GEF-H1 activity revealed that autoinhibited GEF-H1 is localized to MTs, while MT depolymerization subadjacent to the cell cortex promotes GEF-H1 activation in an ~5-µm-wide peripheral band. GEF-H1 is further regulated by Src phosphorylation, activating GEF-H1 in a narrower band ~0-2 µm from the cell edge, in coordination with cell protrusions. This indicates a synergistic intersection between MT dynamics and Src signaling in RhoA activation through GEF-H1.
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http://dx.doi.org/10.1083/jcb.201812073DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6719461PMC
September 2019

An image-based assay to quantify changes in proliferation and viability upon drug treatment in 3D microenvironments.

BMC Cancer 2019 May 28;19(1):502. Epub 2019 May 28.

Department of Cell Biology, UT Southwestern Medical Center, Dallas, TX, USA.

Background: Every biological experiment requires a choice of throughput balanced against physiological relevance. Most primary drug screens neglect critical parameters such as microenvironmental conditions, cell-cell heterogeneity, and specific readouts of cell fate for the sake of throughput.

Methods: Here we describe a methodology to quantify proliferation and viability of single cells in 3D culture conditions by leveraging automated microscopy and image analysis to facilitate reliable and high-throughput measurements. We detail experimental conditions that can be adjusted to increase either throughput or robustness of the assay, and we provide a stand alone image analysis program for users who wish to implement this 3D drug screening assay in high throughput.

Results: We demonstrate this approach by evaluating a combination of RAF and MEK inhibitors on melanoma cells, showing that cells cultured in 3D collagen-based matrices are more sensitive than cells grown in 2D culture, and that cell proliferation is much more sensitive than cell viability. We also find that cells grown in 3D cultured spheroids exhibit equivalent sensitivity to single cells grown in 3D collagen, suggesting that for the case of melanoma, a 3D single cell model may be equally effective for drug identification as 3D spheroids models. The single cell resolution of this approach enables stratification of heterogeneous populations of cells into differentially responsive subtypes upon drug treatment, which we demonstrate by determining the effect of RAK/MEK inhibition on melanoma cells co-cultured with fibroblasts. Furthermore, we show that spheroids grown from single cells exhibit dramatic heterogeneity to drug response, suggesting that heritable drug resistance can arise stochastically in single cells but be retained by subsequent generations.

Conclusion: In summary, image-based analysis renders cell fate detection robust, sensitive, and high-throughput, enabling cell fate evaluation of single cells in more complex microenvironmental conditions.
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http://dx.doi.org/10.1186/s12885-019-5694-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6537405PMC
May 2019

Augmin accumulation on long-lived microtubules drives amplification and kinetochore-directed growth.

J Cell Biol 2019 07 21;218(7):2150-2168. Epub 2019 May 21.

Institute of Molecular Biotechnology of the Austrian Academy of Sciences, Vienna BioCenter, Vienna, Austria

Dividing cells reorganize their microtubule cytoskeleton into a bipolar spindle, which moves one set of sister chromatids to each nascent daughter cell. Early spindle assembly models postulated that spindle pole-derived microtubules search the cytoplasmic space until they randomly encounter a kinetochore to form a stable attachment. More recent work uncovered several additional, centrosome-independent microtubule generation pathways, but the contributions of each pathway to spindle assembly have remained unclear. Here, we combined live microscopy and mathematical modeling to show that most microtubules nucleate at noncentrosomal regions in dividing human cells. Using a live-cell probe that selectively labels aged microtubule lattices, we demonstrate that the distribution of growing microtubule plus ends can be almost entirely explained by Augmin-dependent amplification of long-lived microtubule lattices. By ultrafast 3D lattice light-sheet microscopy, we observed that this mechanism results in a strong directional bias of microtubule growth toward individual kinetochores. Our systematic quantification of spindle dynamics reveals highly coordinated microtubule growth during kinetochore fiber assembly.
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http://dx.doi.org/10.1083/jcb.201805044DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6605806PMC
July 2019

Enhanced Dendritic Actin Network Formation in Extended Lamellipodia Drives Proliferation in Growth-Challenged Rac1 Melanoma Cells.

Dev Cell 2019 05;49(3):444-460.e9

Lyda Hill Department of Bioinformatics, University of Texas Southwestern Medical Center, Dallas, TX 75390, USA; Department of Cell Biology, University of Texas Southwestern Medical Center, Dallas, TX 75390, USA. Electronic address:

Actin assembly supplies the structural framework for cell morphology and migration. Beyond structure, this actin framework can also be engaged to drive biochemical signaling programs. Here, we describe how the hyperactivation of Rac1 via the P29S mutation (Rac1) in melanoma hijacks branched actin network assembly to coordinate proliferative cues that facilitate metastasis and drug resistance. Upon growth challenge, Rac1-harboring melanoma cells massively upregulate lamellipodia formation by dendritic actin polymerization. These extended lamellipodia form a signaling microdomain that sequesters and phospho-inactivates the tumor suppressor NF2/Merlin, driving Rac1 cell proliferation in growth suppressive conditions. These biochemically active lamellipodia require cell-substrate attachment but not focal adhesion assembly and drive proliferation independently of the ERK/MAPK pathway. These data suggest a critical link between cell morphology and cell signaling and reconcile the dichotomy of Rac1's regulation of both proliferation and actin assembly by revealing a mutual signaling axis wherein actin assembly drives proliferation in melanoma.
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http://dx.doi.org/10.1016/j.devcel.2019.04.007DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6760970PMC
May 2019

Nuclear positioning facilitates amoeboid migration along the path of least resistance.

Nature 2019 04 3;568(7753):546-550. Epub 2019 Apr 3.

Institute of Science and Technology Austria (IST Austria), Klosterneuburg, Austria.

During metazoan development, immune surveillance and cancer dissemination, cells migrate in complex three-dimensional microenvironments. These spaces are crowded by cells and extracellular matrix, generating mazes with differently sized gaps that are typically smaller than the diameter of the migrating cell. Most mesenchymal and epithelial cells and some-but not all-cancer cells actively generate their migratory path using pericellular tissue proteolysis. By contrast, amoeboid cells such as leukocytes use non-destructive strategies of locomotion, raising the question how these extremely fast cells navigate through dense tissues. Here we reveal that leukocytes sample their immediate vicinity for large pore sizes, and are thereby able to choose the path of least resistance. This allows them to circumnavigate local obstacles while effectively following global directional cues such as chemotactic gradients. Pore-size discrimination is facilitated by frontward positioning of the nucleus, which enables the cells to use their bulkiest compartment as a mechanical gauge. Once the nucleus and the closely associated microtubule organizing centre pass the largest pore, cytoplasmic protrusions still lingering in smaller pores are retracted. These retractions are coordinated by dynamic microtubules; when microtubules are disrupted, migrating cells lose coherence and frequently fragment into migratory cytoplasmic pieces. As nuclear positioning in front of the microtubule organizing centre is a typical feature of amoeboid migration, our findings link the fundamental organization of cellular polarity to the strategy of locomotion.
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http://dx.doi.org/10.1038/s41586-019-1087-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7217284PMC
April 2019

Automated profiling of growth cone heterogeneity defines relations between morphology and motility.

J Cell Biol 2019 01 6;218(1):350-379. Epub 2018 Dec 6.

Departments of Bioinformatics and Cell Biology, University of Texas Southwestern Medical Center, Dallas, TX

Growth cones are complex, motile structures at the tip of an outgrowing neurite. They often exhibit a high density of filopodia (thin actin bundles), which complicates the unbiased quantification of their morphologies by software. Contemporary image processing methods require extensive tuning of segmentation parameters, require significant manual curation, and are often not sufficiently adaptable to capture morphology changes associated with switches in regulatory signals. To overcome these limitations, we developed Growth Cone Analyzer (GCA). GCA is designed to quantify growth cone morphodynamics from time-lapse sequences imaged both in vitro and in vivo, but is sufficiently generic that it may be applied to nonneuronal cellular structures. We demonstrate the adaptability of GCA through the analysis of growth cone morphological variation and its relation to motility in both an unperturbed system and in the context of modified Rho GTPase signaling. We find that perturbations inducing similar changes in neurite length exhibit underappreciated phenotypic nuance at the scale of the growth cone.
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http://dx.doi.org/10.1083/jcb.201711023DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6314545PMC
January 2019

Profiling cellular morphodynamics by spatiotemporal spectrum decomposition.

PLoS Comput Biol 2018 08 2;14(8):e1006321. Epub 2018 Aug 2.

Dept. of Bioinformatics, University of Texas Southwestern Medical Center, Dallas, Texas, United States of America.

Cellular morphology and associated morphodynamics are widely used for qualitative and quantitative assessments of cell state. Here we implement a framework to profile cellular morphodynamics based on an adaptive decomposition of local cell boundary motion into instantaneous frequency spectra defined by the Hilbert-Huang transform (HHT). Our approach revealed that spontaneously migrating cells with approximately homogeneous molecular makeup show remarkably consistent instantaneous frequency distributions, though they have markedly heterogeneous mobility. Distinctions in cell edge motion between these cells are captured predominantly by differences in the magnitude of the frequencies. We found that acute photo-inhibition of Vav2 guanine exchange factor, an activator of the Rho family of signaling proteins coordinating cell motility, produces significant shifts in the frequency distribution, but does not affect frequency magnitude. We therefore concluded that the frequency spectrum encodes the wiring of the molecular circuitry that regulates cell boundary movements, whereas the magnitude captures the activation level of the circuitry. We also used HHT spectra as multi-scale spatiotemporal features in statistical region merging to identify subcellular regions of distinct motion behavior. In line with our conclusion that different HHT spectra relate to different signaling regimes, we found that subcellular regions with different morphodynamics indeed exhibit distinct Rac1 activities. This algorithm thus can serve as an accurate and sensitive classifier of cellular morphodynamics to pinpoint spatial and temporal boundaries between signaling regimes.
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http://dx.doi.org/10.1371/journal.pcbi.1006321DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6091976PMC
August 2018

Inhibition of epithelial cell migration and Src/FAK signaling by SIRT3.

Proc Natl Acad Sci U S A 2018 07 18;115(27):7057-7062. Epub 2018 Jun 18.

Department of Cell Biology, Harvard Medical School, Boston, MA 02115;

Metastasis remains the leading cause of cancer mortality, and reactive oxygen species (ROS) signaling promotes the metastatic cascade. However, the molecular pathways that control ROS signaling relevant to metastasis are little studied. Here, we identify SIRT3, a mitochondrial deacetylase, as a regulator of cell migration via its control of ROS signaling. We find that, although mitochondria are present at the leading edge of migrating cells, SIRT3 expression is down-regulated during migration, resulting in elevated ROS levels. This SIRT3-mediated control of ROS represses Src oxidation and attenuates focal adhesion kinase (FAK) activation. SIRT3 overexpression inhibits migration and metastasis in breast cancer cells. Finally, in human breast cancers, SIRT3 expression is inversely correlated with metastatic outcome and Src/FAK signaling. Our results reveal a role for SIRT3 in cell migration, with important implications for breast cancer progression.
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http://dx.doi.org/10.1073/pnas.1800440115DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6142214PMC
July 2018

A noncanonical role for dynamin-1 in regulating early stages of clathrin-mediated endocytosis in non-neuronal cells.

PLoS Biol 2018 04 18;16(4):e2005377. Epub 2018 Apr 18.

Department of Cell Biology, UT Southwestern Medical Center, Dallas, Texas, United States of America.

Dynamin Guanosine Triphosphate hydrolases (GTPases) are best studied for their role in the terminal membrane fission process of clathrin-mediated endocytosis (CME), but they have also been proposed to regulate earlier stages of CME. Although highly enriched in neurons, dynamin-1 (Dyn1) is, in fact, widely expressed along with Dyn2 but inactivated in non-neuronal cells via phosphorylation by glycogen synthase kinase-3 beta (GSK3β) kinase. Here, we study the differential, isoform-specific functions of Dyn1 and Dyn2 as regulators of CME. Endogenously expressed Dyn1 and Dyn2 were fluorescently tagged either separately or together in two cell lines with contrasting Dyn1 expression levels. By quantitative live cell dual- and triple-channel total internal reflection fluorescence microscopy, we find that Dyn2 is more efficiently recruited to clathrin-coated pits (CCPs) than Dyn1, and that Dyn2 but not Dyn1 exhibits a pronounced burst of assembly, presumably into supramolecular collar-like structures that drive membrane scission and clathrin-coated vesicle (CCV) formation. Activation of Dyn1 by acute inhibition of GSK3β results in more rapid endocytosis of transferrin receptors, increased rates of CCP initiation, and decreased CCP lifetimes but did not significantly affect the extent of Dyn1 recruitment to CCPs. Thus, activated Dyn1 can regulate early stages of CME that occur well upstream of fission, even when present at low, substoichiometric levels relative to Dyn2. Under physiological conditions, Dyn1 is activated downstream of epidermal growth factor receptor (EGFR) signaling to alter CCP dynamics. We identify sorting nexin 9 (SNX9) as a preferred binding partner to activated Dyn1 that is partially required for Dyn1-dependent effects on early stages of CCP maturation. Together, we decouple regulatory and scission functions of dynamins and report a scission-independent, isoform-specific regulatory role for Dyn1 in CME.
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http://dx.doi.org/10.1371/journal.pbio.2005377DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5927468PMC
April 2018

Regulation of Clathrin-Mediated Endocytosis.

Annu Rev Biochem 2018 06 16;87:871-896. Epub 2018 Apr 16.

Department of Cell Biology, University of Texas Southwestern Medical Center, Dallas, Texas 75390, USA; email: , , , ,

Clathrin-mediated endocytosis (CME) is the major endocytic pathway in mammalian cells. It is responsible for the uptake of transmembrane receptors and transporters, for remodeling plasma membrane composition in response to environmental changes, and for regulating cell surface signaling. CME occurs via the assembly and maturation of clathrin-coated pits that concentrate cargo as they invaginate and pinch off to form clathrin-coated vesicles. In addition to the major coat proteins, clathrin triskelia and adaptor protein complexes, CME requires a myriad of endocytic accessory proteins and phosphatidylinositol lipids. CME is regulated at multiple steps-initiation, cargo selection, maturation, and fission-and is monitored by an endocytic checkpoint that induces disassembly of defective pits. Regulation occurs via posttranslational modifications, allosteric conformational changes, and isoform and splice-variant differences among components of the CME machinery, including the GTPase dynamin. This review summarizes recent findings on the regulation of CME and the evolution of this complex process.
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http://dx.doi.org/10.1146/annurev-biochem-062917-012644DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6383209PMC
June 2018

Discovery of functional interactions among actin regulators by analysis of image fluctuations in an unperturbed motile cell system.

Philos Trans R Soc Lond B Biol Sci 2018 05;373(1747)

Department of Cell Biology, Lyda Hill Department of Bioinformatics, UT Southwestern Medical Center, Dallas, TX 75390, USA.

Cell migration is driven by propulsive forces derived from polymerizing actin that pushes and extends the plasma membrane. The underlying actin network is constantly undergoing adaptation to new mechano-chemical environments and intracellular conditions. As such, mechanisms that regulate actin dynamics inherently contain multiple feedback loops and redundant pathways. Given the highly adaptable nature of such a system, studies that use only perturbation experiments (e.g. knockdowns, overexpression, pharmacological activation/inhibition, etc.) are challenged by the nonlinearity and redundancy of the pathway. In these pathway configurations, perturbation experiments at best describe the function(s) of a molecular component in an adapting (e.g. acutely drug-treated) or fully adapted (e.g. permanent gene silenced) cell system, where the targeted component now resides in a non-native equilibrium. Here, we propose how quantitative live-cell imaging and analysis of constitutive fluctuations of molecular activities can overcome these limitations. We highlight emerging actin filament barbed-end biology as a prime example of a complex, nonlinear molecular process that requires a fluctuation analytic approach, especially in an unperturbed cellular system, to decipher functional interactions of barbed-end regulators, actin polymerization and membrane protrusion.This article is part of the theme issue 'Self-organization in cell biology'.
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http://dx.doi.org/10.1098/rstb.2017.0110DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5904296PMC
May 2018

Piecewise-Stationary Motion Modeling and Iterative Smoothing to Track Heterogeneous Particle Motions in Dense Environments.

IEEE Trans Image Process 2017 Nov;26(11):5395-5410

One of the major challenges in multiple particle tracking is the capture of extremely heterogeneous movements of objects in crowded scenes. The presence of numerous assignment candidates in the expected range of particle motion makes the tracking ambiguous and induces false positives. Lowering the ambiguity by reducing the search range, on the other hand, is not an option, as this would increase the rate of false negatives. We propose here a piecewise-stationary motion model (PMM) for the particle transport along an iterative smoother that exploits recursive tracking in multiple rounds in forward and backward temporal directions. By fusing past and future information, our method, termed PMMS, can recover fast transitions from freely or confined diffusive to directed motions with linear time complexity. To avoid false positives, we complemented recursive tracking with a robust inline estimator of the search radius for assignment (a.k.a. gating), where past and future information are exploited using only two frames at each optimization step. We demonstrate the improvement of our technique on simulated data especially the impact of density, variation in frame to frame displacements, and motion switching probability. We evaluated our technique on the 2D particle tracking challenge dataset published by Chenouard et al. in 2014. Using high SNR to focus on motion modeling challenges, we show superior performance at high particle density. On biological applications, our algorithm allows us to quantify the extremely small percentage of motor-driven movements of fluorescent particles along microtubules in a dense field of unbound, diffusing particles. We also show with virus imaging that our algorithm can cope with a strong reduction in recording frame rate while keeping the same performance relative to methods relying on fast sampling.
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http://dx.doi.org/10.1109/TIP.2017.2707803DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5796444PMC
November 2017

A method for measuring the distribution of the shortest telomeres in cells and tissues.

Nat Commun 2017 11 7;8(1):1356. Epub 2017 Nov 7.

Department of Cell Biology, University of Texas Southwestern Medical Center, 5323 Harry Hines Boulevard, Dallas, TX, 75390, USA.

Improved methods to measure the shortest (not just average) telomere lengths (TLs) are needed. We developed Telomere Shortest Length Assay (TeSLA), a technique that detects telomeres from all chromosome ends from <1 kb to 18 kb using small amounts of input DNA. TeSLA improves the specificity and efficiency of TL measurements that is facilitated by user friendly image-processing software to automatically detect and annotate band sizes, calculate average TL, as well as the percent of the shortest telomeres. Compared with other TL measurement methods, TeSLA provides more information about the shortest telomeres. The length of telomeres was measured longitudinally in peripheral blood mononuclear cells during human aging, in tissues during colon cancer progression, in telomere-related diseases such as idiopathic pulmonary fibrosis, as well as in mice and other organisms. The results indicate that TeSLA is a robust method that provides a better understanding of the shortest length of telomeres.
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http://dx.doi.org/10.1038/s41467-017-01291-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5676791PMC
November 2017

Multiplexed Exchange-PAINT imaging reveals ligand-dependent EGFR and Met interactions in the plasma membrane.

Sci Rep 2017 09 22;7(1):12150. Epub 2017 Sep 22.

HMS LINCS Center, Laboratory of Systems Pharmacology, Harvard Medical School, Boston, MA, 02115, USA.

Signal transduction by receptor tyrosine kinases (RTKs) involves complex ligand- and time-dependent changes in conformation and modification state. High resolution structures are available for individual receptors dimers, but less is known about receptor clusters that form in plasma membranes composed of many different RTKs with the potential to interact. We report the use of multiplexed super-resolution imaging (Exchange-PAINT) followed by mean-shift clustering and random forest analysis to measure the precise distributions of five receptor tyrosine kinases (RTKs) from the ErbB, IGF-1R and Met families in breast cancer cells. We find that these receptors are intermixed nonhomogenously on the plasma membrane. Stimulation by EGF does not appear to induce a change in the density of EGFR in local clusters but instead results in formation of EGFR-Met and EGFR-ErbB3 associations; non-canonical EGFR-Met interactions are implicated in resistance to anti-cancer drugs but have not been previously detected in drug-naïve cells.
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http://dx.doi.org/10.1038/s41598-017-12257-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5610318PMC
September 2017

Cell forces meet cell metabolism.

Nat Cell Biol 2017 May;19(6):591-593

Department of Cell Biology, Lyda Hill Department of Bioinformatics, UT Southwestern Medical Center, Dallas, Texas 75390, USA.

Epithelial cells form energetically costly cell-cell adhesions in response to mechanical forces. How cells obtain their energy during this event is unclear. Activity of a key regulator of cell metabolism, the AMP-activated protein kinase (AMPK), is now shown to be mechanoresponsive, and thus can bridge adhesion mechanotransduction and energy homeostasis.
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http://dx.doi.org/10.1038/ncb3542DOI Listing
May 2017

Diverse roles of guanine nucleotide exchange factors in regulating collective cell migration.

J Cell Biol 2017 06 16;216(6):1543-1556. Epub 2017 May 16.

Cell Biology Program, Memorial Sloan-Kettering Cancer Center, New York, NY 10065.

Efficient collective migration depends on a balance between contractility and cytoskeletal rearrangements, adhesion, and mechanical cell-cell communication, all controlled by GTPases of the RHO family. By comprehensive screening of guanine nucleotide exchange factors (GEFs) in human bronchial epithelial cell monolayers, we identified GEFs that are required for collective migration at large, such as SOS1 and β-PIX, and RHOA GEFs that are implicated in intercellular communication. Down-regulation of the latter GEFs differentially enhanced front-to-back propagation of guidance cues through the monolayer and was mirrored by down-regulation of RHOA expression and myosin II activity. Phenotype-based clustering of knockdown behaviors identified RHOA-ARHGEF18 and ARHGEF3-ARHGEF28-ARHGEF11 clusters, indicating that the latter may signal through other RHO-family GTPases. Indeed, knockdown of RHOC produced an intermediate between the two phenotypes. We conclude that for effective collective migration, the RHOA-GEFs → RHOA/C → actomyosin pathways must be optimally tuned to compromise between generation of motility forces and restriction of intercellular communication.
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http://dx.doi.org/10.1083/jcb.201609095DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5461017PMC
June 2017

Vimentin fibers orient traction stress.

Proc Natl Acad Sci U S A 2017 05 2;114(20):5195-5200. Epub 2017 May 2.

Department of Cell Biology, Harvard Medical School, Boston, MA 02115;

The intermediate filament vimentin is required for cells to transition from the epithelial state to the mesenchymal state and migrate as single cells; however, little is known about the specific role of vimentin in the regulation of mesenchymal migration. Vimentin is known to have a significantly greater ability to resist stress without breaking in vitro compared with actin or microtubules, and also to increase cell elasticity in vivo. Therefore, we hypothesized that the presence of vimentin could support the anisotropic mechanical strain of single-cell migration. To study this, we fluorescently labeled vimentin with an mEmerald tag using TALEN genome editing. We observed vimentin architecture in migrating human foreskin fibroblasts and found that network organization varied from long, linear bundles, or "fibers," to shorter fragments with a mesh-like organization. We developed image analysis tools employing steerable filtering and iterative graph matching to characterize the fibers embedded in the surrounding mesh. Vimentin fibers were aligned with fibroblast branching and migration direction. The presence of the vimentin network was correlated with 10-fold slower local actin retrograde flow rates, as well as spatial homogenization of actin-based forces transmitted to the substrate. Vimentin fibers coaligned with and were required for the anisotropic orientation of traction stresses. These results indicate that the vimentin network acts as a load-bearing superstructure capable of integrating and reorienting actin-based forces. We propose that vimentin's role in cell motility is to govern the alignment of traction stresses that permit single-cell migration.
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http://dx.doi.org/10.1073/pnas.1614610114DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5441818PMC
May 2017