Publications by authors named "Torsten Wittmann"

49 Publications

pHLARE: a new biosensor reveals decreased lysosome pH in cancer cells.

Mol Biol Cell 2021 01 25;32(2):131-142. Epub 2020 Nov 25.

Department of Cell and Tissue Biology, University of California, San Francisco, San Francisco, CA 94941.

Many lysosome functions are determined by a lumenal pH of ∼5.0, including the activity of resident acid-activated hydrolases. Lysosome pH (pHlys) is often increased in neurodegenerative disorders and predicted to be decreased in cancers, making it a potential target for therapeutics to limit the progression of these diseases. Accurately measuring pHlys, however, is limited by currently used dyes that accumulate in multiple intracellular compartments and cannot be propagated in clonal cells for longitudinal studies or used for in vivo determinations. To resolve this limitation, we developed a genetically encoded ratiometric pHlys biosensor, pHLARE (ysosomal ctivity porter), which localizes predominantly in lysosomes, has a dynamic range of pH 4.0 to 6.5, and can be stably expressed in cells. Using pHLARE we show decreased pHlys with inhibiting activity of the mammalian target of rapamycin complex 1 (mTORC1). Also, cancer cells from different tissue origins have a lower pHlys than untransformed cells, and stably expressing oncogenic RasV12 in untransformed cells is sufficient to decrease pHlys. pHLARE is a new tool to accurately measure pHlys for improved understanding of lysosome dynamics, which is increasingly considered a therapeutic target.
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http://dx.doi.org/10.1091/mbc.E20-06-0383DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8120692PMC
January 2021

Neurotoxic microglia promote TDP-43 proteinopathy in progranulin deficiency.

Nature 2020 12 31;588(7838):459-465. Epub 2020 Aug 31.

Department of Pathology, University of California San Francisco, San Francisco, CA, USA.

Aberrant aggregation of the RNA-binding protein TDP-43 in neurons is a hallmark of frontotemporal lobar degeneration caused by haploinsufficiency in the gene encoding progranulin. However, the mechanism leading to TDP-43 proteinopathy remains unclear. Here we use single-nucleus RNA sequencing to show that progranulin deficiency promotes microglial transition from a homeostatic to a disease-specific state that causes endolysosomal dysfunction and neurodegeneration in mice. These defects persist even when Grn microglia are cultured ex vivo. In addition, single-nucleus RNA sequencing reveals selective loss of excitatory neurons at disease end-stage, which is characterized by prominent nuclear and cytoplasmic TDP-43 granules and nuclear pore defects. Remarkably, conditioned media from Grn microglia are sufficient to promote TDP-43 granule formation, nuclear pore defects and cell death in excitatory neurons via the complement activation pathway. Consistent with these results, deletion of the genes encoding C1qa and C3 mitigates microglial toxicity and rescues TDP-43 proteinopathy and neurodegeneration. These results uncover previously unappreciated contributions of chronic microglial toxicity to TDP-43 proteinopathy during neurodegeneration.
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http://dx.doi.org/10.1038/s41586-020-2709-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7746606PMC
December 2020

Lights, cytoskeleton, action: Optogenetic control of cell dynamics.

Curr Opin Cell Biol 2020 10 1;66:1-10. Epub 2020 May 1.

Department of Cell Biology, Erasmus MC, Rotterdam, the Netherlands. Electronic address:

Cell biology is moving from observing molecules to controlling them in real time, a critical step towards a mechanistic understanding of how cells work. Initially developed from light-gated ion channels to control neuron activity, optogenetics now describes any genetically encoded protein system designed to accomplish specific light-mediated tasks. Recent photosensitive switches use many ingenious designs that bring spatial and temporal control within reach for almost any protein or pathway of interest. This next generation optogenetics includes light-controlled protein-protein interactions and shape-shifting photosensors, which in combination with live microscopy enable acute modulation and analysis of dynamic protein functions in living cells. We provide a brief overview of various types of optogenetic switches. We then discuss how diverse approaches have been used to control cytoskeleton dynamics with light through Rho GTPase signaling, microtubule and actin assembly, mitotic spindle positioning and intracellular transport and highlight advantages and limitations of different experimental strategies.
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http://dx.doi.org/10.1016/j.ceb.2020.03.003DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7577957PMC
October 2020

Optogenetic Control of Microtubule Dynamics.

Methods Mol Biol 2020 ;2101:211-234

Department of Cell and Tissue Biology, University of California San Francisco, San Francisco, CA, USA.

Light can be controlled with high spatial and temporal accuracy. Therefore, optogenetics is an attractive experimental approach to modulate intracellular cytoskeleton dynamics at much faster timescales than by genetic modification. For example, in mammalian cells, microtubules (MTs) grow tens of micrometers per minute and many intracellular MT functions are mediated by a complex of +TIP proteins that dynamically associate with growing MT plus ends. EB1 is a central component of this +TIP protein network, and we recently developed a photo-inactivated π-EB1 by inserting a blue light-sensitive LOV2/Zdk1 module between the EB1 MT-binding domain and the +TIP adaptor domain. Blue light-induced π-EB1 photodissociation results in disassembly of the +TIP complex and strongly attenuates MT growth in mammalian cells.In this chapter, we discuss theoretical and practical aspects of how to perform high-resolution live-cell microscopy in combination with π-EB1 photodissociation. However, these techniques are broadly applicable to other LOV2-based and likely other blue light-sensitive optogenetics. In addition to being a tool to investigate +TIP functions acutely and with subcellular resolution, because of its dramatic and rapid change in intracellular localization, π-EB1 can serve as a powerful tool to test and characterize optogenetic illumination setups. We describe protocols on how to achieve micrometer-scale intracellular control of π-EB1 activity using patterned illumination, and we introduce a do-it-yourself LED cube design compatible with transmitted light microscopy in multiwell plates.
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http://dx.doi.org/10.1007/978-1-0716-0219-5_14DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7278073PMC
January 2021

Tau repeat regions contain conserved histidine residues that modulate microtubule-binding in response to changes in pH.

J Biol Chem 2019 05 16;294(22):8779-8790. Epub 2019 Apr 16.

From the Department of Cell and Tissue Biology, University of California San Francisco, San Francisco, California 94143,

Tau, a member of the MAP2/tau family of microtubule-associated proteins, stabilizes and organizes axonal microtubules in healthy neurons. In neurodegenerative tauopathies, tau dissociates from microtubules and forms neurotoxic extracellular aggregates. MAP2/tau family proteins are characterized by three to five conserved, intrinsically disordered repeat regions that mediate electrostatic interactions with the microtubule surface. Here, we used molecular dynamics, microtubule-binding experiments, and live-cell microscopy, revealing that highly-conserved histidine residues near the C terminus of each microtubule-binding repeat are pH sensors that can modulate tau-microtubule interaction strength within the physiological intracellular pH range. We observed that at low pH (<7.5), these histidines are positively charged and interact with phenylalanine residues in a hydrophobic cleft between adjacent tubulin dimers. At higher pH (>7.5), tau deprotonation decreased binding to microtubules both and in cells. Electrostatic and hydrophobic characteristics of histidine were both required for tau-microtubule binding, as substitutions with constitutively and positively charged nonaromatic lysine or uncharged alanine greatly reduced or abolished tau-microtubule binding. Consistent with these findings, tau-microtubule binding was reduced in a cancer cell model with increased intracellular pH but was rapidly restored by decreasing the pH to normal levels. These results add detailed insights into the intracellular regulation of tau activity that may be relevant in both normal and pathological conditions.
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http://dx.doi.org/10.1074/jbc.RA118.007004DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6552421PMC
May 2019

Microtubule Plus End Dynamics - Do We Know How Microtubules Grow?: Cells boost microtubule growth by promoting distinct structural transitions at growing microtubule ends.

Bioessays 2019 03 7;41(3):e1800194. Epub 2019 Feb 7.

Department of Cell and Tissue Biology, University of California San Francisco, 513 Parnassus Avenue, San Francisco, CA, 94143, USA.

Microtubules form a highly dynamic filament network in all eukaryotic cells. Individual microtubules grow by tubulin dimer subunit addition and frequently switch between phases of growth and shortening. These unique dynamics are powered by GTP hydrolysis and drive microtubule network remodeling, which is central to eukaryotic cell biology and morphogenesis. Yet, our knowledge of the molecular events at growing microtubule ends remains incomplete. Here, recent ultrastructural, biochemical and cell biological data are integrated to develop a realistic model of growing microtubule ends comprised of structurally distinct but biochemically overlapping zones. Proteins that recognize microtubule lattice conformations associated with specific tubulin guanosine nucleotide states may independently control major structural transitions at growing microtubule ends. A model is proposed in which tubulin dimer addition and subsequent closure of the MT wall are optimized in cells to achieve rapid physiological microtubule growth.
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http://dx.doi.org/10.1002/bies.201800194DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7021488PMC
March 2019

Local control of intracellular microtubule dynamics by EB1 photodissociation.

Nat Cell Biol 2018 03 29;20(3):252-261. Epub 2018 Jan 29.

Department of Cell and Tissue Biology, University of California, San Francisco, CA, USA.

End-binding proteins (EBs) are adaptors that recruit functionally diverse microtubule plus-end-tracking proteins (+TIPs) to growing microtubule plus ends. To test with high spatial and temporal accuracy how, when and where +TIP complexes contribute to dynamic cell biology, we developed a photo-inactivated EB1 variant (π-EB1) by inserting a blue-light-sensitive protein-protein interaction module between the microtubule-binding and +TIP-binding domains of EB1. π-EB1 replaces endogenous EB1 function in the absence of blue light. By contrast, blue-light-mediated π-EB1 photodissociation results in rapid +TIP complex disassembly, and acutely and reversibly attenuates microtubule growth independent of microtubule end association of the microtubule polymerase CKAP5 (also known as ch-TOG and XMAP215). Local π-EB1 photodissociation allows subcellular control of microtubule dynamics at the second and micrometre scale, and elicits aversive turning of migrating cancer cells. Importantly, light-mediated domain splitting can serve as a template to optically control other intracellular protein activities.
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http://dx.doi.org/10.1038/s41556-017-0028-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5826794PMC
March 2018

The glycolytic enzyme phosphofructokinase-1 assembles into filaments.

J Cell Biol 2017 08 23;216(8):2305-2313. Epub 2017 Jun 23.

Department of Cell and Tissue Biology, University of California, San Francisco, San Francisco, CA

Despite abundant knowledge of the regulation and biochemistry of glycolytic enzymes, we have limited understanding on how they are spatially organized in the cell. Emerging evidence indicates that nonglycolytic metabolic enzymes regulating diverse pathways can assemble into polymers. We now show tetramer- and substrate-dependent filament assembly by phosphofructokinase-1 (PFK1), which is considered the "gatekeeper" of glycolysis because it catalyzes the step committing glucose to breakdown. Recombinant liver PFK1 (PFKL) isoform, but not platelet PFK1 (PFKP) or muscle PFK1 (PFKM) isoforms, assembles into filaments. Negative-stain electron micrographs reveal that filaments are apolar and made of stacked tetramers oriented with exposed catalytic sites positioned along the edge of the polymer. Electron micrographs and biochemical data with a PFKL/PFKP chimera indicate that the PFKL regulatory domain mediates filament assembly. Quantified live-cell imaging shows dynamic properties of localized PFKL puncta that are enriched at the plasma membrane. These findings reveal a new behavior of a key glycolytic enzyme with insights on spatial organization and isoform-specific glucose metabolism in cells.
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http://dx.doi.org/10.1083/jcb.201701084DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5551713PMC
August 2017

GSK3-mediated CLASP2 phosphorylation modulates kinetochore dynamics.

J Cell Sci 2017 04 23;130(8):1404-1412. Epub 2017 Feb 23.

Department of Cell & Tissue Biology, University of California San Francisco, 513 Parnassus Avenue, San Francisco, CA 94143, USA

Error-free chromosome segregation requires dynamic control of microtubule attachment to kinetochores, but how kinetochore-microtubule interactions are spatially and temporally controlled during mitosis remains incompletely understood. In addition to the NDC80 microtubule-binding complex, other proteins with demonstrated microtubule-binding activities localize to kinetochores. One such protein is the cytoplasmic linker-associated protein 2 (CLASP2). Here, we show that global GSK3-mediated phosphorylation of the longest isoform, CLASP2α, largely abolishes CLASP2α-microtubule association in metaphase. However, it does not directly control localization of CLASP2α to kinetochores. Using dominant phosphorylation-site variants, we find that CLASP2α phosphorylation weakens kinetochore-microtubule interactions as evidenced by decreased tension between sister kinetochores. Expression of CLASP2α phosphorylation-site mutants also resulted in increased chromosome segregation defects, indicating that GSK3-mediated control of CLASP2α-microtubule interactions contributes to correct chromosome dynamics. Because of global inhibition of CLASP2α-microtubule interactions, we propose a model in which only kinetochore-bound CLASP2α is dephosphorylated, locally engaging its microtubule-binding activity.
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http://dx.doi.org/10.1242/jcs.194662DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5399784PMC
April 2017

Autophagy in adhesion and migration.

J Cell Sci 2016 10 26;129(20):3685-3693. Epub 2016 Sep 26.

Department of Pathology and Helen Diller Family Comprehensive Cancer Center, University of California San Francisco, San Francisco, CA 94143, USA

Autophagy, a pathway for lysosomal-mediated cellular degradation, has recently been described as a regulator of cell migration. Although the molecular mechanisms underlying autophagy-dependent motility are only beginning to emerge, new work demonstrates that selective autophagy mediated by the autophagy cargo receptor, NBR1, specifically promotes the dynamic turnover of integrin-based focal adhesion sites during motility. Here, we discuss the detailed mechanisms through which NBR1-dependent selective autophagy supports focal adhesion remodeling, and we describe the interconnections between this pathway and other established regulators of focal adhesion turnover, such as microtubules. We also highlight studies that examine the contribution of autophagy to selective degradation of proteins that mediate cellular tension and to integrin trafficking; these findings hint at further roles for autophagy in supporting adhesion and migration. Given the recently appreciated importance of selective autophagy in diverse cellular processes, we propose that further investigation into autophagy-mediated focal adhesion turnover will not only shed light onto how focal adhesions are regulated but will also unveil new mechanisms regulating selective autophagy.
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http://dx.doi.org/10.1242/jcs.188490DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5087656PMC
October 2016

Doublecortin Is Excluded from Growing Microtubule Ends and Recognizes the GDP-Microtubule Lattice.

Curr Biol 2016 06 26;26(12):1549-1555. Epub 2016 May 26.

Department of Cell and Tissue Biology, University of California San Francisco, 513 Parnassus Avenue, San Francisco, CA 94143, USA. Electronic address:

Many microtubule (MT) functions are mediated by a diverse class of proteins (+TIPs) at growing MT plus ends that control intracellular MT interactions and dynamics and depend on end-binding proteins (EBs) [1]. Cryoelectron microscopy has recently identified the EB binding site as the interface of four tubulin dimers that undergoes a conformational change in response to β-tubulin GTP hydrolysis [2, 3]. Doublecortin (DCX), a MT-associated protein (MAP) required for neuronal migration during cortical development [4, 5], binds to the same site as EBs [6], and recent in vitro studies proposed DCX localization to growing MT ends independent of EBs [7]. Because this conflicts with observations in neurons [8, 9] and the molecular function of DCX is not well understood, we revisited intracellular DCX dynamics at low expression levels. Here, we report that DCX is not a +TIP in cells but, on the contrary, is excluded from the EB1 domain. In addition, we find that DCX-MT interactions are highly sensitive to MT geometry. In cells, DCX binding was greatly reduced at MT segments with high local curvature. Remarkably, this geometry-dependent binding to MTs was completely reversed in the presence of taxanes, which reconciles incompatible observations in cells [9] and in vitro [10]. We propose a model explaining DCX specificity for different MT geometries based on structural changes induced by GTP hydrolysis that decreases the spacing between adjacent tubulin dimers [11]. Our data are consistent with a unique mode of MT interaction in which DCX specifically recognizes this compacted GDP-like MT lattice.
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http://dx.doi.org/10.1016/j.cub.2016.04.020DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5023073PMC
June 2016

NBR1 enables autophagy-dependent focal adhesion turnover.

J Cell Biol 2016 Feb 22;212(5):577-90. Epub 2016 Feb 22.

Department of Pathology and Helen Diller Family Comprehensive Cancer Center, University of California, San Francisco, San Francisco, CA 94143

Autophagy is a catabolic pathway involving the sequestration of cellular contents into a double-membrane vesicle, the autophagosome. Although recent studies have demonstrated that autophagy supports cell migration, the underlying mechanisms remain unknown. Using live-cell imaging, we uncover that autophagy promotes optimal migratory rate and facilitates the dynamic assembly and disassembly of cell-matrix focal adhesions (FAs), which is essential for efficient motility. Additionally, our studies reveal that autophagosomes associate with FAs primarily during disassembly, suggesting autophagy locally facilitates the destabilization of cell-matrix contact sites. Furthermore, we identify the selective autophagy cargo receptor neighbor of BRCA1 (NBR1) as a key mediator of autophagy-dependent FA remodeling. NBR1 depletion impairs FA turnover and decreases targeting of autophagosomes to FAs, whereas ectopic expression of autophagy-competent, but not autophagy-defective, NBR1 enhances FA disassembly and reduces FA lifetime during migration. Our findings provide mechanistic insight into how autophagy promotes migration by revealing a requirement for NBR1-mediated selective autophagy in enabling FA disassembly in motile cells.
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http://dx.doi.org/10.1083/jcb.201503075DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4772495PMC
February 2016

Migration of Founder Epithelial Cells Drives Proper Molar Tooth Positioning and Morphogenesis.

Dev Cell 2015 Dec;35(6):713-24

Department of Orofacial Sciences and Program in Craniofacial Biology, University of California, San Francisco, 513 Parnassus Avenue, San Francisco, CA 94143, USA; Department of Pediatrics and Institute for Human Genetics, University of California, San Francisco, 513 Parnassus Avenue, San Francisco, CA 94143, USA. Electronic address:

The proper positioning of organs during development is essential, yet little is known about the regulation of this process in mammals. Using murine tooth development as a model, we have found that cell migration plays a central role in positioning of the organ primordium. By combining lineage tracing, genetic cell ablation, and confocal live imaging, we identified a migratory population of Fgf8-expressing epithelial cells in the embryonic mandible. These Fgf8-expressing progenitors furnish the epithelial cells required for tooth development, and the progenitor population migrates toward a Shh-expressing region in the mandible, where the tooth placode will initiate. Inhibition of Fgf and Shh signaling disrupted the oriented migration of cells, leading to a failure of tooth development. These results demonstrate the importance of intraepithelial cell migration in proper positioning of an initiating organ.
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http://dx.doi.org/10.1016/j.devcel.2015.11.025DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4710359PMC
December 2015

Enzymatically Generated CRISPR Libraries for Genome Labeling and Screening.

Dev Cell 2015 Aug 23;34(3):373-8. Epub 2015 Jul 23.

Department of Molecular and Cell Biology, University of California at Berkeley, Berkeley, CA 94720-3200, USA. Electronic address:

CRISPR-based technologies have emerged as powerful tools to alter genomes and mark chromosomal loci, but an inexpensive method for generating large numbers of RNA guides for whole genome screening and labeling is lacking. Using a method that permits library construction from any source of DNA, we generated guide libraries that label repetitive loci or a single chromosomal locus in Xenopus egg extracts and show that a complex library can target the E. coli genome at high frequency.
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http://dx.doi.org/10.1016/j.devcel.2015.06.003DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4536113PMC
August 2015

Acetylcholine receptor (AChR) clustering is regulated both by glycogen synthase kinase 3β (GSK3β)-dependent phosphorylation and the level of CLIP-associated protein 2 (CLASP2) mediating the capture of microtubule plus-ends.

J Biol Chem 2014 Oct 17;289(44):30857-30867. Epub 2014 Sep 17.

Department of Cell Biology, Erasmus Medical Center, 3015 GE, Rotterdam, Netherlands,. Electronic address:

The postsynaptic apparatus of the neuromuscular junction (NMJ) traps and anchors acetylcholine receptors (AChRs) at high density at the synapse. We have previously shown that microtubule (MT) capture by CLASP2, a MT plus-end-tracking protein (+TIP), increases the size and receptor density of AChR clusters at the NMJ through the delivery of AChRs and that this is regulated by a pathway involving neuronal agrin and several postsynaptic kinases, including GSK3. Phosphorylation by GSK3 has been shown to cause CLASP2 dissociation from MT ends, and nine potential phosphorylation sites for GSK3 have been mapped on CLASP2. How CLASP2 phosphorylation regulates MT capture at the NMJ and how this controls the size of AChR clusters are not yet understood. To examine this, we used myotubes cultured on agrin patches that induce AChR clustering in a two-dimensional manner. We show that expression of a CLASP2 mutant, in which the nine GSK3 target serines are mutated to alanine (CLASP2-9XS/9XA) and are resistant to GSK3β-dependent phosphorylation, promotes MT capture at clusters and increases AChR cluster size, compared with myotubes that express similar levels of wild type CLASP2 or that are noninfected. Conversely, myotubes expressing a phosphomimetic form of CLASP2 (CLASP2-8XS/D) show enrichment of immobile mutant CLASP2 in clusters, but MT capture and AChR cluster size are reduced. Taken together, our data suggest that both GSK3β-dependent phosphorylation and the level of CLASP2 play a role in the maintenance of AChR cluster size through the regulated capture and release of MT plus-ends.
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http://dx.doi.org/10.1074/jbc.M114.589457DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4215261PMC
October 2014

Quantitative imaging in cell biology. Preface.

Methods Cell Biol 2014 ;123:xix-xx

University of California, San Francisco.

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http://dx.doi.org/10.1016/B978-0-12-420138-5.09983-3DOI Listing
February 2015

Analysis of focal adhesion turnover: a quantitative live-cell imaging example.

Methods Cell Biol 2014 ;123:335-46

Department of Cell and Tissue Biology, University of California, San Francisco, USA.

Recent advances in optical and fluorescent protein technology have rapidly raised expectations in cell biology, allowing quantitative insights into dynamic intracellular processes like never before. However, quantitative live-cell imaging comes with many challenges including how best to translate dynamic microscopy data into numerical outputs that can be used to make meaningful comparisons rather than relying on representative data sets. Here, we use analysis of focal adhesion turnover dynamics as a straightforward specific example on how to image, measure, and analyze intracellular protein dynamics, but we believe this outlines a thought process and can provide guidance on how to understand dynamic microcopy data of other intracellular structures.
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http://dx.doi.org/10.1016/B978-0-12-420138-5.00018-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4198331PMC
February 2015

Fluorescence live cell imaging.

Methods Cell Biol 2014 ;123:77-94

Department of Cell and Tissue Biology, University of California, San Francisco, USA.

Fluorescence microscopy of live cells has become an integral part of modern cell biology. Fluorescent protein (FP) tags, live cell dyes, and other methods to fluorescently label proteins of interest provide a range of tools to investigate virtually any cellular process under the microscope. The two main experimental challenges in collecting meaningful live cell microscopy data are to minimize photodamage while retaining a useful signal-to-noise ratio and to provide a suitable environment for cells or tissues to replicate physiological cell dynamics. This chapter aims to give a general overview on microscope design choices critical for fluorescence live cell imaging that apply to most fluorescence microscopy modalities and on environmental control with a focus on mammalian tissue culture cells. In addition, we provide guidance on how to design and evaluate FP constructs by spinning disk confocal microscopy.
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http://dx.doi.org/10.1016/B978-0-12-420138-5.00005-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4198327PMC
February 2015

Concepts in quantitative fluorescence microscopy.

Methods Cell Biol 2014 ;123:1-18

Department of Cell and Tissue Biology, University of California, San Francisco, USA.

In recent years, there has been an enormous increase in the publication of spatial and temporal measurements made on fluorescence microscopy digital images. Quantitative fluorescence microscopy is a powerful and important tool in biological research but is also an error-prone technique that requires careful attention to detail. In this chapter, we focus on general concepts that are critical to performing accurate and precise quantitative fluorescence microscopy measurements.
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http://dx.doi.org/10.1016/B978-0-12-420138-5.00001-XDOI Listing
February 2015

Neuronal deletion of GSK3β increases microtubule speed in the growth cone and enhances axon regeneration via CRMP-2 and independently of MAP1B and CLASP2.

BMC Biol 2014 Jun 12;12:47. Epub 2014 Jun 12.

Background: In the adult central nervous system, axonal regeneration is abortive. Regulators of microtubule dynamics have emerged as attractive targets to promote axonal growth following injury as microtubule organization is pivotal for growth cone formation. In this study, we used conditioned neurons with high regenerative capacity to further dissect cytoskeletal mechanisms that might be involved in the gain of intrinsic axon growth capacity.

Results: Following a phospho-site broad signaling pathway screen, we found that in conditioned neurons with high regenerative capacity, decreased glycogen synthase kinase 3β (GSK3β) activity and increased microtubule growth speed in the growth cone were present. To investigate the importance of GSK3β regulation during axonal regeneration in vivo, we used three genetic mouse models with high, intermediate or no GSK3β activity in neurons. Following spinal cord injury, reduced GSK3β levels or complete neuronal deletion of GSK3β led to increased growth cone microtubule growth speed and promoted axon regeneration. While several microtubule-interacting proteins are GSK3β substrates, phospho-mimetic collapsin response mediator protein 2 (T/D-CRMP-2) was sufficient to decrease microtubule growth speed and neurite outgrowth of conditioned neurons and of GSK3β-depleted neurons, prevailing over the effect of decreased levels of phosphorylated microtubule-associated protein 1B (MAP1B) and through a mechanism unrelated to decreased levels of phosphorylated cytoplasmic linker associated protein 2 (CLASP2). In addition, phospho-resistant T/A-CRMP-2 counteracted the inhibitory myelin effect on neurite growth, further supporting the GSK3β-CRMP-2 relevance during axon regeneration.

Conclusions: Our work shows that increased microtubule growth speed in the growth cone is present in conditions of increased axonal growth, and is achieved following inactivation of the GSK3β-CRMP-2 pathway, enhancing axon regeneration through the glial scar. In this context, our results support that a precise control of microtubule dynamics, specifically in the growth cone, is required to optimize axon regrowth.
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http://dx.doi.org/10.1186/1741-7007-12-47DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4229956PMC
June 2014

CLASPs link focal-adhesion-associated microtubule capture to localized exocytosis and adhesion site turnover.

Nat Cell Biol 2014 Jun 25;16(6):561-73. Epub 2014 May 25.

Department of Cell and Tissue Biology, University of California San Francisco, 513 Parnassus Avenue San Francisco, California 94143, USA.

Turnover of integrin-based focal adhesions (FAs) with the extracellular matrix (ECM) is essential for coordinated cell movement. In collectively migrating human keratinocytes, FAs assemble near the leading edge, grow and mature as a result of contractile forces and disassemble underneath the advancing cell body. We report that clustering of microtubule-associated CLASP1 and CLASP2 proteins around FAs temporally correlates with FA turnover. CLASPs and LL5β (also known as PHLDB2), which recruits CLASPs to FAs, facilitate FA disassembly. CLASPs are further required for FA-associated ECM degradation, and matrix metalloprotease inhibition slows FA disassembly similarly to CLASP or PHLDB2 (LL5β) depletion. Finally, CLASP-mediated microtubule tethering at FAs establishes an FA-directed transport pathway for delivery, docking and localized fusion of exocytic vesicles near FAs. We propose that CLASPs couple microtubule organization, vesicle transport and cell interactions with the ECM, establishing a local secretion pathway that facilitates FA turnover by severing cell-matrix connections.
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http://dx.doi.org/10.1038/ncb2975DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4108447PMC
June 2014

Host cell polarity proteins participate in innate immunity to Pseudomonas aeruginosa infection.

Cell Host Microbe 2014 May;15(5):636-43

Division of Infectious Disease, Department of Medicine, University of California, San Francisco, San Francisco, CA 94143, USA; Department of Microbiology and Immunology, University of California, San Francisco, San Francisco, CA 94143, USA. Electronic address:

The mucosal epithelium consists of polarized cells with distinct apical and basolateral membranes that serve as functional and physical barriers to external pathogens. The apical surface of the epithelium constitutes the first point of contact between mucosal pathogens, such as Pseudomonas aeruginosa, and their host. We observed that binding of P. aeruginosa aggregates to the apical surface of polarized cells led to the striking formation of an actin-rich membrane protrusion with inverted polarity, containing basolateral lipids and membrane components. Such protrusions were associated with a spatially localized host immune response to P. aeruginosa aggregates that required bacterial flagella and a type III secretion system apparatus. Host protrusions formed de novo underneath bacterial aggregates and involved the apical recruitment of a Par3/Par6α/aPKC/Rac1 signaling module for a robust, spatially localized host NF-κB response. Our data reveal a role for spatiotemporal epithelial polarity changes in the activation of innate immune responses.
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http://dx.doi.org/10.1016/j.chom.2014.04.007DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4062193PMC
May 2014

Expression of an expanded CGG-repeat RNA in a single pair of primary sensory neurons impairs olfactory adaptation in Caenorhabditis elegans.

Hum Mol Genet 2014 Sep 12;23(18):4945-59. Epub 2014 May 12.

Department of Biochemistry and Molecular Medicine, University of California, Davis, School of Medicine, Davis, CA 95616, USA, MIND Institute, University of California, Davis, Health System, Sacramento, CA 95817, USA

Fragile X-associated tremor/ataxia syndrome (FXTAS) is a severe neurodegenerative disorder that affects carriers of premutation CGG-repeat expansion alleles of the fragile X mental retardation 1 (FMR1) gene; current evidence supports a causal role of the expanded CGG repeat within the FMR1 mRNA in the pathogenesis of FXTAS. Though the mRNA has been observed to induce cellular toxicity in FXTAS, the mechanisms are unclear. One common neurophysiological characteristic of FXTAS patients is their inability to properly attenuate their response to an auditory stimulus upon receipt of a small pre-stimulus. Therefore, to gain genetic and cell biological insight into FXTAS, we examined the effect of expanded CGG repeats on the plasticity of the olfactory response of the genetically tractable nematode, Caenorhabditis elegans (C. elegans). While C. elegans is innately attracted to odors, this response can be downregulated if the odor is paired with starvation. We found that expressing expanded CGG repeats in olfactory neurons interfered with this plasticity without affecting either the innate odor-seeking response or the olfactory neuronal morphology. Interrogation of three RNA regulatory pathways indicated that the expanded CGG repeats act via the C. elegans microRNA (miRNA)-specific Argonaute ALG-2 to diminish olfactory plasticity. This observation suggests that the miRNA-Argonaute pathway may play a pathogenic role in subverting neuronal function in FXTAS.
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http://dx.doi.org/10.1093/hmg/ddu210DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4140470PMC
September 2014

LIS1 controls mitosis and mitotic spindle organization via the LIS1-NDEL1-dynein complex.

Hum Mol Genet 2014 Jan 12;23(2):449-66. Epub 2013 Sep 12.

Department of Pediatrics, Institute for Human Genetics.

Heterozygous LIS1 mutations are responsible for the human neuronal migration disorder lissencephaly. Mitotic functions of LIS1 have been suggested from many organisms throughout evolution. However, the cellular functions of LIS1 at distinct intracellular compartments such as the centrosome and the cell cortex have not been well defined especially during mitotic cell division. Here, we used detailed cellular approaches and time-lapse live cell imaging of mitosis from Lis1 mutant mouse embryonic fibroblasts to reveal critical roles of LIS1 in mitotic spindle regulation. We found that LIS1 is required for the tight control of chromosome congression and segregation to dictate kinetochore-microtubule (MT) interactions and anaphase progression. In addition, LIS1 is essential for the establishment of mitotic spindle pole integrity by maintaining normal centrosome number. Moreover, LIS1 plays crucial roles in mitotic spindle orientation by increasing the density of astral MT plus-end movements toward the cell cortex, which enhances cortical targeting of LIS1-dynein complex. Overexpression of NDEL1-dynein and MT stabilization rescues spindle orientation defects in Lis1 mutants, demonstrating that mouse LIS1 acts via the LIS1-NDEL1-dynein complex to regulate astral MT plus-ends dynamics and establish proper contacts of MTs with the cell cortex to ensure precise cell division.
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http://dx.doi.org/10.1093/hmg/ddt436DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3869364PMC
January 2014

Microtubule dynamics regulation contributes to endothelial morphogenesis.

Bioarchitecture 2012 Nov-Dec;2(6):220-7

Department of Cell and Tissue Biology, University of California at San Francisco, USA.

Because little is known how microtubules contribute to cell migration in a physiological three-dimensional environment, we analyzed microtubule function and dynamics during in vitro angiogenesis in which endothelial cells form networks on a reconstituted basement membrane. Endothelial network formation resulted from distinct cell behaviors: matrix reorganization by myosin-mediated contractile forces, and active cell migration along reorganized, bundled matrix fibers. Inhibition of microtubule dynamics inhibited persistent cell migration, but not matrix reorganization. In addition, microtubule polymerization dynamics and CLASP2-binding to microtubules were spatially regulated to promote microtubule growth into endothelial cell protrusions along matrix tension tracks. We propose that microtubules counter-act contractile forces of the cortical actin cytoskeleton and are required to stabilize endothelial cell protrusions in a soft three-dimensional environment.
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http://dx.doi.org/10.4161/bioa.22335DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3527317PMC
January 2014

Host microtubule plus-end binding protein CLASP1 influences sequential steps in the Trypanosoma cruzi infection process.

Cell Microbiol 2013 Apr 20;15(4):571-84. Epub 2012 Nov 20.

Department of Immunology and Infectious Disease, Harvard School of Public Health, 665 Huntington Ave, Boston, MA, 02115, USA.

Mammalian cell invasion by the protozoan parasite Trypanosoma cruzi involves host cell microtubule dynamics. Microtubules support kinesin-dependent anterograde trafficking of host lysosomes to the cell periphery where targeted lysosome exocytosis elicits remodelling of the plasma membrane and parasite invasion. Here, a novel role for microtubule plus-end tracking proteins (+TIPs) in the co-ordination of T. cruzi trypomastigote internalization and post-entry events is reported. Acute silencing of CLASP1, a +TIP that participates in microtubule stabilization at the cell periphery, impairs trypomastigote internalization without diminishing the capacity for calcium-regulated lysosome exocytosis. Subsequent fusion of the T. cruzi vacuole with host lysosomes and its juxtanuclear positioning are also delayed in CLASP1-depleted cells. These post-entry phenotypes correlate with a generalized impairment of minus-end directed transport of lysosomes in CLASP1 knock-down cells and mimic the effects of dynactin disruption. Consistent with GSK3β acting as a negative regulator of CLASP function, inhibition of GSK3β activity enhances T. cruzi entry in a CLASP1-dependent manner and expression of constitutively active GSK3β dampens infection. This study provides novel molecular insights into the T. cruzi infection process, emphasizing functional links between parasite-elicited signalling, host microtubule plus-end tracking proteins and dynein-based retrograde transport. Highlighted in this work is a previously unrecognized role for CLASPs in dynamic lysosome positioning, an important aspect of the nutrient sensing response in mammalian cells.
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http://dx.doi.org/10.1111/cmi.12056DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3582772PMC
April 2013

Targeting and transport: how microtubules control focal adhesion dynamics.

J Cell Biol 2012 Aug;198(4):481-9

Department of Cell and Tissue Biology, University of California, San Francisco, San Francisco, CA 94143, USA.

Directional cell migration requires force generation that relies on the coordinated remodeling of interactions with the extracellular matrix (ECM), which is mediated by integrin-based focal adhesions (FAs). Normal FA turnover requires dynamic microtubules, and three members of the diverse group of microtubule plus-end-tracking proteins are principally involved in mediating microtubule interactions with FAs. Microtubules also alter the assembly state of FAs by modulating Rho GTPase signaling, and recent evidence suggests that microtubule-mediated clathrin-dependent and -independent endocytosis regulates FA dynamics. In addition, FA-associated microtubules may provide a polarized microtubule track for localized secretion of matrix metalloproteases (MMPs). Thus, different aspects of the molecular mechanisms by which microtubules control FA turnover in migrating cells are beginning to emerge.
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http://dx.doi.org/10.1083/jcb.201206050DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3514042PMC
August 2012

+TIPs: SxIPping along microtubule ends.

Trends Cell Biol 2012 Aug 28;22(8):418-28. Epub 2012 Jun 28.

Department of Cell and Tissue Biology, University of California San Francisco, 513 Parnassus Avenue, San Francisco, CA 94143, USA.

+TIPs are a heterogeneous class of proteins that specifically bind to growing microtubule ends. Because dynamic microtubules are essential for many intracellular processes, +TIPs play important roles in regulating microtubule dynamics and microtubule interactions with other intracellular structures. End-binding proteins (EBs) recognize a structural cap at growing microtubule ends, and have emerged as central adaptors that mediate microtubule plus-end tracking of potentially all other +TIPs. The majority of these +TIPs bind to EBs through a short hydrophobic (S/T)x(I/L)P sequence motif (SxIP) and surrounding electrostatic interactions. These recent discoveries have resulted in a rapid expansion of the number of possible +TIPs. In this review, we outline our current understanding of the molecular mechanism of plus-end tracking and provide an overview of SxIP-recruited +TIPs.
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http://dx.doi.org/10.1016/j.tcb.2012.05.005DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3408562PMC
August 2012

EB1-recruited microtubule +TIP complexes coordinate protrusion dynamics during 3D epithelial remodeling.

Curr Biol 2012 May 5;22(9):753-62. Epub 2012 Apr 5.

Department of Cell and Tissue Biology, University of California, San Francisco, San Francisco, CA 94143, USA.

Background: Epithelial remodeling, in which apical-basal polarized cells switch to a migratory phenotype, plays a central role in development and disease of multicellular organisms. Although dynamic microtubules (MTs) are required for directed migration on flat surfaces, how MT dynamics are controlled or contribute to epithelial remodeling in a more physiological three-dimensional (3D) environment is not understood. We use confocal live-cell imaging to analyze MT function and dynamics during 3D epithelial morphogenesis and remodeling of polarized Madin-Darby canine kidney epithelial cells that undergo partial epithelial-to-mesenchymal transition in response to hepatocyte growth factor (HGF).

Results: We find that HGF treatment increases MT growth rate before morphological changes are evident and that large numbers of MTs grow into HGF-induced cell extensions independent of centrosome reorientation. Using lentivirus-mediated small hairpin RNA, we demonstrate that EB1, an adaptor protein that mediates recruitment of numerous other +TIP proteins to growing MT plus ends, is required for this HGF-induced MT reorganization. We further show that protrusion and adhesion dynamics are disorganized and that vesicular trafficking to the tip of HGF-induced cell extensions is disrupted in EB1-depleted cells.

Conclusions: We conclude that EB1-mediated interactions with growing MTs are important to coordinate cell shape changes and directed migration into the surrounding extracellular matrix during epithelial remodeling in a physiological 3D environment. In contrast, EB1 is not required for the establishment or maintenance of apical-basal cell polarity, suggesting different functions of +TIPs and MTs in different types of cell polarity.
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http://dx.doi.org/10.1016/j.cub.2012.02.069DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3350573PMC
May 2012

Multisite phosphorylation disrupts arginine-glutamate salt bridge networks required for binding of cytoplasmic linker-associated protein 2 (CLASP2) to end-binding protein 1 (EB1).

J Biol Chem 2012 May 29;287(21):17050-17064. Epub 2012 Mar 29.

Department of Cell and Tissue Biology, University of California, San Francisco, California 94143. Electronic address:

A group of diverse proteins reversibly binds to growing microtubule plus ends through interactions with end-binding proteins (EBs). These +TIPs control microtubule dynamics and microtubule interactions with other intracellular structures. Here, we use cytoplasmic linker-associated protein 2 (CLASP2) binding to EB1 to determine how multisite phosphorylation regulates interactions with EB1. The central, intrinsically disordered region of vertebrate CLASP proteins contains two SXIP EB1 binding motifs that are required for EB1-mediated plus-end-tracking in vitro. In cells, both EB1 binding motifs can be functional, but most of the binding free energy results from nearby electrostatic interactions. By employing molecular dynamics simulations of the EB1 interaction with a minimal CLASP2 plus-end-tracking module, we find that conserved arginine residues in CLASP2 form extensive hydrogen-bond networks with glutamate residues predominantly in the unstructured, acidic C-terminal tail of EB1. Multisite phosphorylation of glycogen synthase kinase 3 (GSK3) sites near the EB1 binding motifs disrupts this electrostatic "molecular Velcro." Molecular dynamics simulations and (31)P NMR spectroscopy indicate that phosphorylated serines participate in intramolecular interactions with and sequester arginine residues required for EB1 binding. Multisite phosphorylation of these GSK3 motifs requires priming phosphorylation by interphase or mitotic cyclin-dependent kinases (CDKs), and we find that CDK- and GSK3-dependent phosphorylation completely disrupts CLASP2 microtubule plus-end-tracking in mitosis.
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http://dx.doi.org/10.1074/jbc.M111.316661DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3366819PMC
May 2012