Publications by authors named "Lynne Cassimeris"

45 Publications

Continuous digital hypothermia reduces expression of keratin 17 and 1L-17A inflammatory pathway mediators in equine laminitis induced by hyperinsulinemia.

Vet Immunol Immunopathol 2021 Nov 21;241:110326. Epub 2021 Sep 21.

382 West Street Rd., Department of Clinical Studies, New Bolton Center, School of Veterinary Medicine, University of Pennsylvania, Kennett Square, PA, 19348, USA.

The euglycemic hyperinsulinemic clamp model (EHC) of equine endocrinopathic laminitis induces rapid loss of lamellar tissue integrity, disrupts keratinocyte functions, and induces inflammation similar to natural disease. Continuous digital hypothermia (CDH) blocks tissue damage in this experimental model, allowing identification of specific genes or molecular pathways contributing to disease initiation or early progression. Archived lamellar tissues (8 horses, 48 h EHC treatment, including CDH-treated front limbs) were used to measure relative expression levels of genes encoding keratin 17 (KRT17), a stress-induced intermediate filament protein, and genes upregulated downstream of keratin 17 and/or interleukin 17A (IL-17A), as mediators of inflammation. Compared to front or hind limbs at ambient temperature, CDH resulted in significantly lower expression of KRT17, CCL2, CxCL8, PTGS2 (encoding COX2), IL6, TNFα, S100A8 and MMP1. By immunofluorescence, COX2 was robustly expressed in lamellar keratinocytes from ambient limbs, but not in CDH-treated limbs. Genes not significantly reduced by CDH were IL17A, DEFB4B, S100A9 and MMP9. Overall, 8 of 12 genes were expressed at lower levels in the CDH-treated limb. These 8 genes are expressed by wounded or stress-activated keratinocytes in human disease or mouse models, highlighting the role of keratinocytes in equine laminitis.
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http://dx.doi.org/10.1016/j.vetimm.2021.110326DOI Listing
November 2021

Interleukin-17A pathway target genes are upregulated in Equus caballus supporting limb laminitis.

PLoS One 2020 10;15(12):e0232920. Epub 2020 Dec 10.

Department of Clinical Studies/New Bolton Center, School of Veterinary Medicine, University of Pennsylvania, Kennett Square, Pennsylvania, United States of America.

Supporting Limb Laminitis (SLL) is a painful and crippling secondary complication of orthopedic injuries and infections in horses, often resulting in euthanasia. SLL causes structural alterations and inflammation of the interdigitating layers of specialized epidermal and dermal tissues, the lamellae, which suspend the equine distal phalanx from the hoof capsule. Activation of the interleukin-17A (IL-17A)-dependent inflammatory pathway is an epidermal stress response that contributes to physiologic cutaneous wound healing as well as pathological skin conditions. As a first test of the hypothesis that hoof lamellae of horses diagnosed with SLL also respond to stress by activating the IL-17A pathway, the expression of IL-17A, IL-17 receptor subunit A and 11 IL-17A effector genes was measured by RT-PCR or qPCR. Lamellar tissue was isolated from Thoroughbreds euthanized due to naturally occurring SLL and in age and breed matched non-laminitic controls. By RT-PCR, the IL-17 Receptor A subunit was expressed in both non-laminitic and laminitic tissues, while IL-17A was primarily detectable in laminitic tissues. IL-17A target gene expression was undetectable in non-laminitic samples with the exception of weak detection of DEFB4B, S100A9 and PTSG2. In contrast, all target genes examined, except CCL20, were expressed by some or all laminitic samples. By qPCR, severe acute (n = 7) SLL expressed ~15-100 fold higher levels of DEFB4B and S100A9 genes compared to non-laminitic controls (n = 8). DEFB4B was also upregulated in developmental/subclinical (n = 8) and moderate acute (n = 7) by ~ 5-fold, and in severe chronic (n = 5) by ~15-200 fold. In situ hybridization (DEFB4) and immunofluorescence (calprotectin, a dimer of S100A9/S100A8 proteins) demonstrated expression in keratinocytes, primarily in suprabasal cell layers, from SLL samples. These data demonstrate upregulation of a cohort of IL-17A target genes in SLL and support the hypothesis that similarities in the response to stresses and damage exist between equine and human epidermal tissues.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0232920PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7728170PMC
January 2021

The expression of equine keratins K42 and K124 is restricted to the hoof epidermal lamellae of Equus caballus.

PLoS One 2019 24;14(9):e0219234. Epub 2019 Sep 24.

Department of Clinical Studies, New Bolton Center, University of Pennsylvania, School of Veterinary Medicine, Kennett Square, Pennsylvania, United States of America.

The equine hoof inner epithelium is folded into primary and secondary epidermal lamellae which increase the dermo-epidermal junction surface area of the hoof and can be affected by laminitis, a common disease of equids. Two keratin proteins (K), K42 and K124, are the most abundant keratins in the hoof lamellar tissue of Equus caballus. We hypothesize that these keratins are lamellar tissue-specific and could serve as differentiation- and disease-specific markers. Our objective was to characterize the expression of K42 and K124 in equine stratified epithelia and to generate monoclonal antibodies against K42 and K124. By RT-PCR analysis, keratin gene (KRT) KRT42 and KRT124 expression was present in lamellar tissue, but not cornea, haired skin, or hoof coronet. In situ hybridization studies showed that KRT124 localized to the suprabasal and, to a lesser extent, basal cells of the lamellae, was absent from haired skin and hoof coronet, and abruptly transitions from KRT124-negative coronet to KRT124-positive proximal lamellae. A monoclonal antibody generated against full-length recombinant equine K42 detected a lamellar keratin of the appropriate size, but also cross-reacted with other epidermal keratins. Three monoclonal antibodies generated against N- and C-terminal K124 peptides detected a band of the appropriate size in lamellar tissue and did not cross-react with proteins from haired skin, corneal limbus, hoof coronet, tongue, glabrous skin, oral mucosa, or chestnut on immunoblots. K124 localized to lamellar cells by indirect immunofluorescence. This is the first study to demonstrate the localization and expression of a hoof lamellar-specific keratin, K124, and to validate anti-K124 monoclonal antibodies.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0219234PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6759161PMC
March 2020

Reorganization of paclitaxel-stabilized microtubule arrays at mitotic entry: roles of depolymerizing kinesins and severing proteins.

Cancer Biol Ther 2019 25;20(10):1337-1347. Epub 2019 Jul 25.

Department of Biological Sciences, 111 Research Dr. Lehigh University , Bethlehem , PA , USA.

Paclitaxel is a widely used anti-cancer treatment that disrupts cell cycle progression by blocking cells in mitosis. The block at mitosis, with spindles assembled from short microtubules, is surprising given paclitaxel's microtubule stabilizing activity and the need to depolymerize long interphase microtubules prior to spindle formation. Cells must antagonize paclitaxel's microtubule stabilizing activity during a brief window of time at the transition from interphase to mitosis, allowing microtubule reorganization into a mitotic spindle, although the mechanism underlying microtubule depolymerization in the presence of paclitaxel has not been examined. Here we test the hypothesis that microtubule severing and/or depolymerizing proteins active at mitotic entry are necessary to clear the interphase array in paclitaxel-treated cells and allow subsequent formation of mitotic spindles formed of short microtubules. A549 and LLC-PK1 cells treated with 30nM paclitaxel approximately 4 h prior to mitotic entry successfully progress through the G2/M transition by clearing the interphase microtubule array from the cell interior outward to the cell periphery, a spatial pattern of reorganization that differs from that of cells possessing dynamic microtubules. Depletion of kinesin-8s, KIF18A and/or KIF18B obstructed interphase microtubule clearing at mitotic entry in paclitaxel-treated cells, with KIF18B making the larger contribution. Of the severing proteins, depletion of spastin, but not katanin, reduced microtubule loss as cells entered mitosis in the presence of paclitaxel. These results support a model in which KIF18A, KIF18B, and spastin promote interphase microtubule array disassembly at mitotic entry and can overcome paclitaxel-induced microtubule stability specifically at the G2/M transition.
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http://dx.doi.org/10.1080/15384047.2019.1638678DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6783116PMC
August 2020

Detection of endoplasmic reticulum stress and the unfolded protein response in naturally-occurring endocrinopathic equine laminitis.

BMC Vet Res 2019 Jan 10;15(1):24. Epub 2019 Jan 10.

Department of Clinical Studies/New Bolton Center, School of Veterinary Medicine, University of Pennsylvania, Kennett Square, PA, 19348, USA.

Background: Laminitis is often associated with endocrinopathies that cause hyperinsulinemia and is also induced experimentally by hyperinsulinemia, suggesting that insulin initiates laminitis pathogenesis. Hyperinsulinemia is expected to activate pro-growth and anabolic signaling pathways. We hypothesize that chronic over-stimulation of these pathways in lamellar tissue results in endoplasmic reticulum stress, contributing to tissue pathology, as it does in human metabolic diseases. We tested this hypothesis by asking whether lamellar tissue from horses with naturally-occurring endocrinopathic laminitis showed expression of protein markers of endoplasmic reticulum stress.

Results: Three markers of endoplasmic reticulum stress, spliced XBP1, Grp78/BiP and Grp94, were upregulated 2.5-9.5 fold in lamellar tissues of moderately to severely laminitic front limbs (n = 12) compared to levels in controls (n = 6-7) measured by immunoblotting and densitometry. Comparing expression levels between laminitic front limbs and less affected hind limbs from the same horses (paired samples from 7 to 8 individual horses) demonstrated significantly higher expression for both spliced XBP1 and Grp78/BiP in the laminitic front limbs, and a similar trend for Grp94. Expression levels of the 3 markers were minimal in all samples of the control (n = 6-7) or hind limb groups (n = 7-8). Immunofluorescent localizations were used to identify cell types expressing high levels of Grp78/BiP, as an indicator of endoplasmic reticulum stress. Grp78/BiP expression was highly elevated in suprabasal epidermal keratinocytes and only observed in laminitic front limbs (10/12 laminitic samples, compared to 0/7 in sections from the hind limbs and 0/5 of controls).

Conclusions: These data demonstrate that the endoplasmic reticulum stress pathway is active in naturally occurring cases of laminitis and is most active within a subset of epidermal keratinocytes. These data provide the rationale for further study of endoplasmic reticulum stress in experimental models of laminitis and the links between laminitis and human diseases sharing activation of this stress pathway. Pharmacological options to manipulate the endoplasmic reticulum stress pathway under investigation for human disease could be applicable to laminitis treatment and prevention should this pathway prove to be a driver of disease progression.
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http://dx.doi.org/10.1186/s12917-018-1748-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6327420PMC
January 2019

Monte Carlo simulations of microtubule arrays: The critical roles of rescue transitions, the cell boundary, and tubulin concentration in shaping microtubule distributions.

PLoS One 2018 21;13(5):e0197538. Epub 2018 May 21.

Dept. of Biomedical Engineering, University of Minnesota, Minneapolis, Minnesota, United States of America.

Microtubules are dynamic polymers required for a number of processes, including chromosome movement in mitosis. While regulators of microtubule dynamics have been well characterized, we lack a convenient way to predict how the measured dynamic parameters shape the entire microtubule system within a cell, or how the system responds when specific parameters change in response to internal or external signals. Here we describe a Monte Carlo model to simulate an array of dynamic microtubules from parameters including the cell radius, total tubulin concentration, microtubule nucleation rate from the centrosome, and plus end dynamic instability. The algorithm also allows dynamic instability or position of the cell edge to vary during the simulation. Outputs from simulations include free tubulin concentration, average microtubule lengths, length distributions, and individual length changes over time. Using this platform and reported parameters measured in interphase LLCPK1 epithelial cells, we predict that sequestering ~ 15-20% of total tubulin results in fewer microtubules, but promotes dynamic instability of those remaining. Simulations also predict that lowering nucleation rate will increase the stability and average length of the remaining microtubules. Allowing the position of the cell's edge to vary over time changed the average length but not the number of microtubules and generated length distributions consistent with experimental measurements. Simulating the switch from interphase to prophase demonstrated that decreased rescue frequency at prophase is the critical factor needed to rapidly clear the cell of interphase microtubules prior to mitotic spindle assembly. Finally, consistent with several previous simulations, our results demonstrate that microtubule nucleation and dynamic instability in a confined space determines the partitioning of tubulin between monomer and polymer pools. The model and simulations will be useful for predicting changes to the entire microtubule array after modification to one or more parameters, including predicting the effects of tubulin-targeted chemotherapies.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0197538PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5962052PMC
December 2018

The chemical biology of Cu(II) complexes with imidazole or thiazole containing ligands: Synthesis, crystal structures and comparative biological activity.

J Inorg Biochem 2016 Apr 11;157:52-61. Epub 2016 Jan 11.

Department of Chemistry and Biochemistry, Union College, Schenectady, NY 12308, United States. Electronic address:

The synthesis and characterization of two copper(II) complexes containing 2-(2-pyridyl)benzimidazole (PyBIm) are reported with the biological activity of these two complexes and a third Cu(II) complex containing 2-(2-pyridyl)benzothiazole (PyBTh). Complex 1, [Cu(PyBIm)(NO3)(H2O)](NO3), is a four coordinate, distorted square planar species with one ligand (N,N), nitrate and water bound to Cu(II). The [Cu(PyBIm)3](BF4)2 complex (2) has distorted octahedral geometry with a 3:1 Py(BIm) ligand to metal ratio. The distorted trigonal bi-pyramidal geometry of compound 3, [Cu(PyBTh)2(H2O)](BF4)2, is comprised of two PyBTh ligands and one water. Biological activity of 1-3 has been assessed by analyzing DNA interaction, nuclease ability, cytotoxic activity and antibacterial properties. Complex 3 exhibits potent concentration dependent SC-DNA cleavage forming single- and double-nicked DNA in contrast to the weak activity of complexes 1 and 2. Mechanistic studies indicate that all complexes utilize an oxidative mechanism however 1 and 2 employ O2(-) as the principal reactive oxygen species while the highly active 3 utilizes (1)O2. The interaction between 1-3 and DNA was investigated using fluorescence emission spectroscopy and revealed all complexes strongly intercalate DNA with Kapp values of 2.65 × 10(6), 1.85 × 10(6) and 2.72 × 10(6)M(-1), respectively. Cytotoxic effects of 1-3 were examined using HeLa and K562 cells and show cell death in the micromolar range with the activity of 1 ≈ 2 and were slightly higher than 3. Similar reactivity was observed in the antibacterial studies with E. coli and S. aureus. A detailed comparative analysis of the three complexes is presented.
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http://dx.doi.org/10.1016/j.jinorgbio.2016.01.014DOI Listing
April 2016

A delay prior to mitotic entry triggers caspase 8-dependent cell death in p53-deficient Hela and HCT-116 cells.

Cell Cycle 2015 ;14(7):1070-81

a Department of Biological Sciences ; Lehigh University ; Bethlehem , PA USA.

Stathmin/Oncoprotein 18, a microtubule destabilizing protein, is required for survival of p53-deficient cells. Stathmin-depleted cells are slower to enter mitosis, but whether delayed mitotic entry triggers cell death or whether stathmin has a separate pro-survival function was unknown. To test these possibilities, we abrogated the cell cycle delay by inhibiting Wee1 in synchronized, stathmin-depleted cells and found that apoptosis was reduced to control levels. Synchronized cells treated with a 4 hour pulse of inhibitors to CDK1 or both Aurora A and PLK1 delayed mitotic entry and apoptosis was triggered only in p53-deficient cells. We did not detect mitotic defects downstream of the delayed mitotic entry, indicating that cell death is activated by a mechanism distinct from those activated by prolonged mitotic arrest. Cell death is triggered by initiator caspase 8, based on its cleavage to the active form and by rescue of viability after caspase 8 depletion or treatment with a caspase 8 inhibitor. In contrast, initiator caspase 9, activated by prolonged mitotic arrest, is not activated and is not required for apoptosis under our experimental conditions. P53 upregulates expression of cFLIPL, a protein that blocks caspase 8 activation. cFLIPL levels are lower in cells lacking p53 and these levels are reduced to a greater extent after stathmin depletion. Expression of FLAG-tagged cFLIPL in p53-deficient cells rescues them from apoptosis triggered by stathmin depletion or CDK1 inhibition during G2. These data indicate that a cell cycle delay in G2 activates caspase 8 to initiate apoptosis specifically in p53-deficient cells.
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http://dx.doi.org/10.1080/15384101.2015.1007781DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4612104PMC
December 2015

Synthesis, characterization, crystal structures and biological activity of set of Cu(II) benzothiazole complexes: artificial nucleases with cytotoxic activities.

J Inorg Biochem 2014 Aug 12;137:1-11. Epub 2014 Apr 12.

Department of Chemistry and Biochemistry, Union College, Schenectady, NY 12308, United States. Electronic address:

A series of Cu(II) complexes with ligand frames based on quinoline derivatives appended with a benzothiazole substituent has been isolated. The complexes, Cu(Q(oBt))(NO3)2(H2O)∙CH3OH (1∙CH3OH), Cu(8OHQ(oBt))Cl2∙CH3OH (2∙CH3OH), Cu(8OQ(oBt))Cl(CH3OH)∙CH3OH (3∙CH3OH) and [Cu(8OH1/2Q(oBt))(CH3OH)(NO3)]2(NO3) (4) have been characterized by single crystal X-ray diffraction, IR and UV-visible spectroscopies, and elemental analysis. The ligand frame within the set of complexes differs in the substituent on the quinoline ring: complex 1 remains unsubstituted at this position while complexes 2-4 have a substituted OH group. In complex 2, the bound phenol remains protonated while in 3 it is a phenolato group. Complex 4 contains two complexes within the unit cell and one NO3(-) giving rise to an overall 'half-protonation'. The interaction between complexes 1-3 with CT-DNA was investigated using fluorescence emission spectroscopy and revealed 2 and 3 strongly intercalate DNA with Kapp values of 1.47×10(7)M(-1) and 3.09×10(7)M(-1), respectively. The ability of complexes 1-3 to cleave SC-DNA was monitored using gel electrophoresis. Each complex exhibits potent, concentration dependent nuclease activity forming single and double-nicked DNA as low as 10μM. The nuclease activity of complexes 1-3 is primarily dependent on (1)O2 species while ·OH radicals play a secondary role in the cleavage by complexes 2 and 3. The cytotoxic effects of 1-3 were examined using HeLa cells and show cell death in the micromolar range. The distribution of cell cycle stages remains unchanged when complexes are present indicating DNA damage may be occurring throughout the cell cycle.
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http://dx.doi.org/10.1016/j.jinorgbio.2014.04.002DOI Listing
August 2014

CAMSAPs add to the growing microtubule minus-end story.

Dev Cell 2014 Feb;28(3):221-2

Department of Biological Sciences, Lehigh University, 111 Research Drive, Bethlehem, PA 18015, USA. Electronic address:

Free microtubule minus ends, found in many differentiated cells, contribute to polarized motility. Work from Jiang et al. (2014) in this issue of Developmental Cell shows how mammalian CAMSAP proteins stabilize minus ends, providing a key piece to the puzzle of how these minus ends are formed and stabilized.
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http://dx.doi.org/10.1016/j.devcel.2014.01.025DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3966109PMC
February 2014

Reversible action of diaminothiazoles in cancer cells is implicated by the induction of a fast conformational change of tubulin and suppression of microtubule dynamics.

Mol Cancer Ther 2014 Jan 5;13(1):179-89. Epub 2013 Nov 5.

Corresponding Author: Suparna Sengupta, Division of Cancer Research, Rajiv Gandhi Centre for Biotechnology, Thiruvananthapuram 695014, India.

Diaminothiazoles are novel cytotoxic compounds that have shown efficacy toward different cancer cell lines. They show potent antimitotic and antiangiogenic activity upon binding to the colchicine-binding site of tubulin. However, the mechanism of action of diaminothiazoles at the molecular level is not known. Here, we show a reversible binding to tubulin with a fast conformational change that allows the lead diaminothiazole DAT1 [4-amino-5-benzoyl-2-(4-methoxy phenyl amino)thiazole] to cause a reversible mitotic block. DAT1 also suppresses microtubule dynamic instability at much lower concentration than its IC(50) value in cancer cells. Both growth and shortening events were reduced by DAT1 in a concentration-dependent way. Colchicine, the long-studied tubulin-binding drug, has previously failed in the treatment of cancer due to its toxicity, even though it generates a strong apoptotic response. The toxicity is attributable to its slow removal from the cell due to irreversible tubulin binding caused by a slow conformational change. DAT1 binds to tubulin at an optimal pH lower than colchicine. Tubulin conformational studies showed that the binding environments of DAT1 and colchicine are different. Molecular dynamic simulations showed a difference in the number of H-bonding interactions that accounts for the different pH optima. This study gives an insight of the action of compounds targeting tubulin's colchicine-binding site, as many such compounds have entered into clinical trials recently.
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http://dx.doi.org/10.1158/1535-7163.MCT-13-0479DOI Listing
January 2014

Stathmin and microtubules regulate mitotic entry in HeLa cells by controlling activation of both Aurora kinase A and Plk1.

Mol Biol Cell 2013 Dec 23;24(24):3819-31. Epub 2013 Oct 23.

Department of Biological Sciences, Lehigh University, Bethlehem, PA 18015.

Depletion of stathmin, a microtubule (MT) destabilizer, delays mitotic entry by ∼4 h in HeLa cells. Stathmin depletion reduced the activity of CDC25 and its upstream activators, Aurora A and Plk1. Chemical inhibition of both Aurora A and Plk1 was sufficient to delay mitotic entry by 4 h, while inhibiting either kinase alone did not cause a delay. Aurora A and Plk1 are likely regulated downstream of stathmin, because the combination of stathmin knockdown and inhibition of Aurora A and Plk1 was not additive and again delayed mitotic entry by 4 h. Aurora A localization to the centrosome required MTs, while stathmin depletion spread its localization beyond that of γ-tubulin, indicating an MT-dependent regulation of Aurora A activation. Plk1 was inhibited by excess stathmin, detected in in vitro assays and cells overexpressing stathmin-cyan fluorescent protein. Recruitment of Plk1 to the centrosome was delayed in stathmin-depleted cells, independent of MTs. It has been shown that depolymerizing MTs with nocodazole abrogates the stathmin-depletion induced cell cycle delay; in this study, depolymerization with nocodazole restored Plk1 activity to near normal levels, demonstrating that MTs also contribute to Plk1 activation. These data demonstrate that stathmin regulates mitotic entry, partially via MTs, to control localization and activation of both Aurora A and Plk1.
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http://dx.doi.org/10.1091/mbc.E13-02-0108DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3861079PMC
December 2013

Specific in vivo labeling of tyrosinated α-tubulin and measurement of microtubule dynamics using a GFP tagged, cytoplasmically expressed recombinant antibody.

PLoS One 2013 28;8(3):e59812. Epub 2013 Mar 28.

Department of Biological Sciences, Lehigh University, Bethlehem, Pennsylvania, United States of America.

GFP-tagged proteins are used extensively as biosensors for protein localization and function, but the GFP moiety can interfere with protein properties. An alternative is to indirectly label proteins using intracellular recombinant antibodies (scFvs), but most antibody fragments are insoluble in the reducing environment of the cytosol. From a synthetic hyperstable human scFv library we isolated an anti-tubulin scFv, 2G4, which is soluble in mammalian cells when expressed as a GFP-fusion protein. Here we report the use of this GFP-tagged scFv to label microtubules in fixed and living cells. We found that 2G4-GFP localized uniformly along microtubules and did not disrupt binding of EB1, a protein that binds microtubule ends and serves as a platform for binding by a complex of proteins regulating MT polymerization. TOGp and CLIP-170 also bound microtubule ends in cells expressing 2G4-GFP. Microtubule dynamic instability, measured by tracking 2G4-GFP labeled microtubules, was nearly identical to that measured in cells expressing GFP-α-tubulin. Fluorescence recovery after photobleaching demonstrated that 2G4-GFP turns over rapidly on microtubules, similar to the turnover rates of fluorescently tagged microtubule-associated proteins. These data indicate that 2G4-GFP binds relatively weakly to microtubules, and this conclusion was confirmed in vitro. Purified 2G4 partially co-pelleted with microtubules, but a significant fraction remained in the soluble fraction, while a second anti-tubulin scFv, 2F12, was almost completely co-pelleted with microtubules. In cells, 2G4-GFP localized to most microtubules, but did not co-localize with those composed of detyrosinated α-tubulin, a post-translational modification associated with non-dynamic, more stable microtubules. Immunoblots probing bacterially expressed tubulins confirmed that 2G4 recognized α-tubulin and required tubulin's C-terminal tyrosine residue for binding. Thus, a recombinant antibody with weak affinity for its substrate can be used as a specific intracellular biosensor that can differentiate between unmodified and post-translationally modified forms of a protein.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0059812PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3610906PMC
January 2014

Dissecting the nanoscale distributions and functions of microtubule-end-binding proteins EB1 and ch-TOG in interphase HeLa cells.

PLoS One 2012 12;7(12):e51442. Epub 2012 Dec 12.

Optical Image Analysis Unit, RIKEN Center for Developmental Biology, Kobe, Hyogo, Japan.

Recently, the EB1 and XMAP215/TOG families of microtubule binding proteins have been demonstrated to bind autonomously to the growing plus ends of microtubules and regulate their behaviour in in vitro systems. However, their functional redundancy or difference in cells remains obscure. Here, we compared the nanoscale distributions of EB1 and ch-TOG along microtubules using high-resolution microscopy techniques, and also their roles in microtubule organisation in interphase HeLa cells. The ch-TOG accumulation sites protruded ∼100 nm from the EB1 comets. Overexpression experiments showed that ch-TOG and EB1 did not interfere with each other's localisation, confirming that they recognise distinct regions at the ends of microtubules. While both EB1 and ch-TOG showed similar effects on microtubule plus end dynamics and additively increased microtubule dynamicity, only EB1 exhibited microtubule-cell cortex attachment activity. These observations indicate that EB1 and ch-TOG regulate microtubule organisation differently via distinct regions in the plus ends of microtubules.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0051442PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3520847PMC
June 2013

The microtubule cytoskeleton is required for a G2 cell cycle delay in cancer cells lacking stathmin and p53.

Cytoskeleton (Hoboken) 2012 May 29;69(5):278-89. Epub 2012 Mar 29.

Department of Biological Sciences, Lehigh University, Bethlehem, Pennsylvania 18015, USA.

In several cancer cell lines, depleting the microtubule (MT)-destabilizing protein stathmin/oncoprotein18 leads to a G2 cell cycle delay and apoptosis. These phenotypes are observed only in synergy with low levels of p53, but the pathway(s) activated by stathmin depletion to delay the cell cycle are unknown. We found that stathmin depletion caused greater MT stability in synergy with loss of p53, measured by the levels of acetylated α-tubulin and the rate of centrosomal MT nucleation. Nocodazole or vinblastine-induced MT depolymerization abrogated the stathmin-depletion induced G2 delay, measured by the percentage of cells staining positive for several markers (TPX2, CDK1 with inhibitory phosphorylation), indicating that MTs are required to lengthen G2. Live cell imaging showed that stathmin depletion increased time in G2 without an impact on the duration of mitosis, indicating that the longer interphase duration is not simply a consequence of a previous slowed mitosis. In contrast, stabilization of MTs with paclitaxel (8 nM) slowed mitosis without lengthening the duration of interphase, demonstrating that increased MT stability alone is not sufficient to delay cells in G2.
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http://dx.doi.org/10.1002/cm.21024DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4004097PMC
May 2012

Fueled by microtubules: does tubulin dimer/polymer partitioning regulate intracellular metabolism?

Cytoskeleton (Hoboken) 2012 Mar 5;69(3):133-43. Epub 2012 Mar 5.

Department of Biological Sciences, Lehigh University, Bethlehem, Pennsylvania 18015, USA.

Microtubules (MTs) or their subunits, tubulin dimers, interact with multiple components that contribute to intracellular metabolic pathways. MTs are required for insulin-dependent transport of glucose transporter 4 to the plasma membrane, they bind most glycolytic enzymes and are required for translation of the mRNA encoding hypoxia inducible factor-1α. Tubulin dimers bind the voltage-dependent anion channel of the mitochondrial outer membrane; this channel functions in metabolite transport in and out of mitochondria. We hypothesize that tubulin partitioning between dimer and polymer pools regulates multiple steps in metabolism, where metabolic output is greatest when both tubulin dimers and MT polymers are present and reduced by drug treatments that disrupt this normal balance. Experimental evidence from these drug-induced changes in tubulin dimer/polymer partitioning supports our model for several metabolic steps. Signal transduction pathways that stabilize or destabilize MTs can shift the normal ratio between unpolymerized and polymerized tubulin dimers, and one downstream consequence of this shift in tubulin partitioning could be a change in metabolic output.
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http://dx.doi.org/10.1002/cm.21008DOI Listing
March 2012

Microtubules: in vivo. Preface.

Methods Cell Biol 2010 ;97:xvii-xviii

Department of Biological Sciences, Lehigh University, Bethlehem, PA, USA.

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http://dx.doi.org/10.1016/S0091-679X(10)97031-3DOI Listing
December 2010

Regulation of microtubule dynamics by Bim1 and Bik1, the budding yeast members of the EB1 and CLIP-170 families of plus-end tracking proteins.

Mol Biol Cell 2010 Jun 14;21(12):2013-23. Epub 2010 Apr 14.

Department of Molecular Biology and Genetics, Cornell University, Ithaca, NY 14853, USA.

Microtubule dynamics are regulated by plus-end tracking proteins (+TIPs), which bind microtubule ends and influence their polymerization properties. In addition to binding microtubules, most +TIPs physically associate with other +TIPs, creating a complex web of interactions. To fully understand how +TIPs regulate microtubule dynamics, it is essential to know the intrinsic biochemical activities of each +TIP and how +TIP interactions affect these activities. Here, we describe the activities of Bim1 and Bik1, two +TIP proteins from budding yeast and members of the EB1 and CLIP-170 families, respectively. We find that purified Bim1 and Bik1 form homodimers that interact with each other to form a tetramer. Bim1 binds along the microtubule lattice but with highest affinity for the microtubule end; however, Bik1 requires Bim1 for localization to the microtubule lattice and end. In vitro microtubule polymerization assays show that Bim1 promotes microtubule assembly, primarily by decreasing the frequency of catastrophes. In contrast, Bik1 inhibits microtubule assembly by slowing growth and, consequently, promoting catastrophes. Interestingly, the Bim1-Bik1 complex affects microtubule dynamics in much the same way as Bim1 alone. These studies reveal new activities for EB1 and CLIP-170 family members and demonstrate how interactions between two +TIP proteins influence their activities.
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http://dx.doi.org/10.1091/mbc.e10-02-0083DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2883945PMC
June 2010

Stathmin/oncoprotein 18, a microtubule regulatory protein, is required for survival of both normal and cancer cell lines lacking the tumor suppressor, p53.

Cancer Biol Ther 2010 May 8;9(9):699-709. Epub 2010 May 8.

Department of Biological Sciences, Lehigh University, Bethlehem, PA, USA.

Stathmin, a microtubule regulatory protein, is overexpressed in many cancers and required for survival of several cancer lines. In a study of breast cancer cell lines(1) proposed that stathmin is required for survival of cells lacking p53, but this hypothesis was not tested directly. Here we tested their hypothesis by examining cell survival in cells depleted of stathmin, p53 or both proteins. Comparing HCT116 colon cancer cell lines differing in TP53 genotype, stathmin depletion resulted in significant death only in cells lacking p53. As a second experimental system, we compared the effects of stathmin depletion from HeLa cells, which normally lack detectable levels of p53 due to expression of the HPV E6 protein. Stathmin depletion caused a large percentage of HeLa cells to die. Restoring p53, by depletion of HPV E6, rescued HeLa cells from stathmin-depletion induced death. Cleaved PARP was detected in HCT116(p53-/-) cells depleted of stathmin and cell death in stathmin-depleted HeLa cells was blocked by the caspase inhibitor Z-VAD-FMK, consistent with apoptotic death. The stathmin-dependent survival of cells lacking p53 was not confined to cancerous cells because both proteins were required for survival of normal human fibroblasts. In HCT116 and HeLa cells, depletion of both stathmin and p53 leads to a cell cycle delay through G(2). Our results demonstrate that stathmin is required for cell survival in cells lacking p53, suggesting that stathmin depletion could be used therapeutically to induce apoptosis in tumors without functional p53.
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http://dx.doi.org/10.4161/cbt.9.9.11430DOI Listing
May 2010

Gene expression profiles in mouse embryo fibroblasts lacking stathmin, a microtubule regulatory protein, reveal changes in the expression of genes contributing to cell motility.

BMC Genomics 2009 Jul 30;10:343. Epub 2009 Jul 30.

Chemistry Department, Lehigh University, Bethlehem, PA 18015, USA.

Background: Stathmin (STMN1) protein functions to regulate assembly of the microtubule cytoskeleton by destabilizing microtubule polymers. Stathmin over-expression has been correlated with cancer stage progression, while stathmin depletion leads to death of some cancer cell lines in culture. In contrast, stathmin-null mice are viable with minor axonopathies and loss of innate fear response. Several stathmin binding partners, in addition to tubulin, have been shown to affect cell motility in culture. To expand our understanding of stathmin function in normal cells, we compared gene expression profiles, measured by microarray and qRT-PCR, of mouse embryo fibroblasts isolated from STMN1+/+ and STMN1-/- mice to determine the transcriptome level changes present in the genetic knock-out of stathmin.

Results: Microarray analysis of STMN1 loss at a fold change threshold of > or = 2.0 revealed expression changes for 437 genes, of which 269 were up-regulated and 168 were down-regulated. Microarray data and qRT-PCR analysis of mRNA expression demonstrated changes in the message levels for STMN4, encoding RB3, a protein related to stathmin, and in alterations to many tubulin isotype mRNAs. KEGG Pathway analysis of the microarray data indicated changes to cell motility-related genes, and qRT-PCR plates specific for focal adhesion and ECM proteins generally confirmed the microarray data. Several microtubule assembly regulators and motors were also differentially regulated in STMN1-/- cells, but these changes should not compensate for loss of stathmin.

Conclusion: Approximately 50% of genes up or down regulated (at a fold change of > or = 2) in STMN1-/- mouse embryo fibroblasts function broadly in cell adhesion and motility. These results support models indicating a role for stathmin in regulating cell locomotion, but also suggest that this functional activity may involve changes to the cohort of proteins expressed in the cell, rather than as a direct consequence of stathmin-dependent regulation of the microtubule cytoskeleton.
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http://dx.doi.org/10.1186/1471-2164-10-343DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2725145PMC
July 2009

Stathmin regulates centrosomal nucleation of microtubules and tubulin dimer/polymer partitioning.

Mol Biol Cell 2009 Aug 10;20(15):3451-8. Epub 2009 Jun 10.

Department of Chemistry and Biological Sciences, Lehigh University, Bethlehem, PA 18015, USA.

Stathmin is a microtubule-destabilizing protein ubiquitously expressed in vertebrates and highly expressed in many cancers. In several cell types, stathmin regulates the partitioning of tubulin between unassembled and polymer forms, but the mechanism responsible for partitioning has not been determined. We examined stathmin function in two cell systems: mouse embryonic fibroblasts (MEFs) isolated from embryos +/+, +/-, and -/- for the stathmin gene and porcine kidney epithelial (LLCPK) cells expressing stathmin-cyan fluorescent protein (CFP) or injected with stathmin protein. In MEFs, the relative amount of stathmin corresponded to genotype, where cells heterozygous for stathmin expressed half as much stathmin mRNA and protein as wild-type cells. Reduction or loss of stathmin resulted in increased microtubule polymer but little change to microtubule dynamics at the cell periphery. Increased stathmin level in LLCPK cells, sufficient to reduce microtubule density, but allowing microtubules to remain at the cell periphery, also did not have a major impact on microtubule dynamics. In contrast, stathmin level had a significant effect on microtubule nucleation rate from centrosomes, where lower stathmin levels increased nucleation and higher stathmin levels reduced nucleation. The stathmin-dependent regulation of nucleation is only active in interphase; overexpression of stathmin-CFP did not impact metaphase microtubule nucleation rate in LLCPK cells and the number of astral microtubules was similar in stathmin +/+ and -/- MEFs. These data support a model in which stathmin functions in interphase to control the partitioning of tubulins between dimer and polymer pools by setting the number of microtubules per cell.
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http://dx.doi.org/10.1091/mbc.e09-02-0140DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2719563PMC
August 2009

TOGp regulates microtubule assembly and density during mitosis and contributes to chromosome directional instability.

Cell Motil Cytoskeleton 2009 Aug;66(8):535-45

Department of Biological Sciences, Lehigh University, Bethlehem, Pennsylvania, USA.

TOGp, a member of the XMAP215 MAP family, is required for bipolar mitotic spindle assembly. To understand how TOGp contributes to spindle assembly, we examined microtubule dynamics after depleting TOGp by siRNA. Fluorescence recovery after photobleaching of GFP-tubulin demonstrated that spindle microtubule turnover is slowed two-fold in the absence of TOGp. Consistent with photobleaching results, microtubule regrowth after washout of the microtubule depolymerizing drug nocodazole was slower at the centrosomes and in the vicinity of mitotic chromatin in cells depleted of TOGp. The slower microtubule turnover is likely due to either nucleation or the transitions of dynamic instability because TOGp depletion did not effect the rate of plus end growth, measured by tracking EB1-GFP at microtubule ends. In contrast, microtubule regrowth after nocodazole washout was unaffected by prior depletion of TACC3, a centrosomal protein that interacts with TOGp. Kinetochore fibers in both untreated and TOGp-depleted cells were stable to incubation at 4 degrees C or lysis in buffer containing calcium indicating that stable kinetochore-microtubule attachments are formed in the absence of TOGp. Depletion of TOGp, but not TACC3, reduced kinetochore oscillations during prometaphase/metaphase. Defects in oscillations are not due simply to multipolarity or loss of centrosome focus in the TOGp-depleted cells, since kinetochore oscillations appear normal in cells treated with the proteosome inhibitor MG132, which also results in multipolar spindles and centrosome fragmentation. We hypothesize that TOGp is required for chromosome motility as a downstream consequence of reduced microtubule dynamics and/or density. Cell Motil. Cytoskeleton 2009. (c) 2009 Wiley-Liss, Inc.
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http://dx.doi.org/10.1002/cm.20359DOI Listing
August 2009

Microtubule assembly: lattice GTP to the rescue.

Authors:
Lynne Cassimeris

Curr Biol 2009 Feb;19(4):R174-6

Department of Biological Sciences, Lehigh University, Bethlehem, PA 18017, USA.

Recent work describes the surprising finding that cellular microtubules have islands of GTP-bound tubulin within their lattices, in contrast to the long-standing view that all but the very tips of growing microtubules are made up entirely of GDP-tubulin. These GTP-tubulin islands may act as stop signs or speed bumps, switching a shortening microtubule back into a growing state.
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http://dx.doi.org/10.1016/j.cub.2008.12.035DOI Listing
February 2009

Positional analyses of BRCA1-dependent expression in Saccharomyces cerevisiae.

Cell Cycle 2008 Dec 10;7(24):3928-34. Epub 2008 Dec 10.

Lehigh University, Department of Biological Sciences, Bethlehem, Pennsylvania 18015, USA.

Mutations in BRCA1 account for a significant proportion of hereditary breast and ovarian cancers, but analysis of BRCA1 function is complicated by pleiotropic effects and binding partners (Pol II holoenzyme and transcription factors, chromatin remodelers, recombination complexes and E3 ligases). In vertebrate cells, efforts to elucidate BRCA1 transcriptional effects have focused on specific genes or restricted portions of the genome-limiting analyses of BRCA1 effects on adjoining DNA sequences and along chromosome lengths. Here, we use microarray analyses on the genetically tractable yeast cell system to elucidate BRCA1-dependent genomewide positional effects on both gene induction and repression. Yeast responses may be of clinical relevance based on findings that BRCA1 severely diminishes yeast growth kinetics but that BRCA1 mutated at sites identified from breast tumors is no longer able to retard yeast cell growth kinetics. Our analysis suggests that BRCA1 acts through both transcription factors to upregulate specific loci and chromatin remodeling complexes to effect global changes in gene expression. BRCA1 also exhibits gene repression activities. Cluster-functional analysis reveals that these repressed factors are required for mitotic stability and provide a novel molecular explanation for the conditional lethality observed between BRCA1 and chromosome segregation genes.
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http://dx.doi.org/10.4161/cc.7.24.7380DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3241008PMC
December 2008

Cellular entry and nuclear targeting by a highly anionic molecular umbrella.

Bioconjug Chem 2008 Aug 6;19(8):1510-3. Epub 2008 Aug 6.

Departments of Chemistry and Biological Sciences, Lehigh University, Bethlehem, Pennsylvania 18015, USA.

A fluorescently labeled, persulfated molecular umbrella ( 1) has been synthesized from cholic acid, lysine, spermine, and Coumarin 343 and found capable of entering live HeLa cells. The distributions of 1 throughout the cytoplasm and the nucleus were diffuse and punctate, respectively. This finding, together with its ability to cross liposomal membranes by passive diffusion, suggests that passive diffusion plays a significant role in the ability of 1 to enter cells. The fact that 1 is concentrated at the nucleus raises the possibility that molecular umbrellas of this type could be used for the nuclear targeting of drugs.
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http://dx.doi.org/10.1021/bc8001826DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2711778PMC
August 2008

The formin mDia2 stabilizes microtubules independently of its actin nucleation activity.

J Cell Biol 2008 May;181(3):523-36

Department of Pathology, Anatomy and Cell Biology, Columbia University, New York, NY 10032, USA.

A critical microtubule (MT) polarization event in cell migration is the Rho/mDia-dependent stabilization of a subset of MTs oriented toward the direction of migration. Although mDia nucleates actin filaments, it is unclear whether this or a separate activity of mDia underlies MT stabilization. We generated two actin mutants (K853A and I704A) in a constitutively active version of mDia2 containing formin homology domains 1 and 2 (FH1FH2) and found that they still induced stable MTs and bound to the MT TIP proteins EB1 and APC, which have also been implicated in MT stabilization. A dimerization-impaired mutant of mDia2 (W630A) also generated stable MTs in cells. We examined whether FH1FH2mDia2 had direct activity on MTs in vitro and found that it bound directly to MTs, stabilized MTs against cold- and dilution-induced disassembly, and reduced the rates of growth and shortening during MT assembly and disassembly, respectively. These results indicate that mDia2 has a novel MT stabilization activity that is separate from its actin nucleation activity.
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http://dx.doi.org/10.1083/jcb.200709029DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2364705PMC
May 2008

Tubulin delivery: polymerization chaperones for microtubule assembly?

Authors:
Lynne Cassimeris

Dev Cell 2007 Oct;13(4):455-6

Department of Biological Sciences, 111 Research Drive, Lehigh University, Bethlehem, PA 18015, USA.

A new paper by Slep and Vale in a recent issue of Molecular Cell provides structural clues as to how three different +TIP proteins interact with tubulin and suggests that +TIPs deliver oligomers of tubulin dimers to growing microtubule ends.
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http://dx.doi.org/10.1016/j.devcel.2007.09.009DOI Listing
October 2007

The contributions of microtubule stability and dynamic instability to adenovirus nuclear localization efficiency.

Cell Motil Cytoskeleton 2007 Sep;64(9):675-89

Department of Biological Sciences, Lehigh University, Bethlehem, Pennsylvania 18015, USA.

Adenoviruses (Ads) utilize host cell microtubules to traverse the intracellular space and reach the nucleus in a highly efficient manner. Previous studies have shown that Ad infection promotes the formation of stable, posttranslationally modified microtubules by a RhoA-dependent mechanism. Ad infection also shifts key parameters of microtubule dynamic instability by a Rac1-dependent mechanism, resulting in microtubules with lower catastrophe frequencies, persistent growth phases, and a bias toward net growth compared to microtubules in uninfected cells. Until now it was unclear whether changes in RhoGTPase activity or microtubule dynamics had a direct impact on the efficiency of Ad microtubule-dependent nuclear localization. Here we have performed synchronous Ad infections and utilized confocal microscopy to analyze the individual contributions of RhoA activation, Rac1 activation, microtubule stability, dynamic behavior, and posttranslational modifications on Ad nuclear localization efficiency (NLE). We found that drug-induced suppression of microtubule dynamics impaired Ad NLE by disrupting the radial organization of the microtubule array. When the microtubule array was maintained, the suppression or enhancement of microtubule turnover did not significantly affect Ad NLE. Furthermore, RhoA activation or the formation of acetylated microtubules did not enhance Ad NLE. In contrast, active Rac1 was required for efficient Ad nuclear localization. Because Rac1 mediates persistent growth of microtubules to the lamellar regions of cells, we propose that Ad-induced activation of Rac1 enhances the ability of microtubules to "search and capture" incoming virus particles.
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http://dx.doi.org/10.1002/cm.20215DOI Listing
September 2007

A novel cancer therapy approach targeting microtubule function.

Cancer Biol Ther 2006 Dec 20;5(12):1721-3. Epub 2006 Dec 20.

Program in Cell and Developmental Biology, Fox Chase Cancer Center, Philadelphia, Pennsylvania 19111, USA.

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http://dx.doi.org/10.4161/cbt.5.12.3736DOI Listing
December 2006

Q & A: Lynne Cassimeris. Interview.

Authors:
Lynne Cassimeris

Curr Biol 2006 Jul;16(13):R480-1

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http://dx.doi.org/10.1016/j.cub.2006.06.015DOI Listing
July 2006
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