The PET and LIM1-2 domains of testin contribute to intramolecular and homodimeric interactions

The focal adhesion protein testin is a modular scaffold and tumour suppressor that consists of an N-terminal cysteine rich (CR) domain, a PET domain of unknown function and three C-terminal LIM domains. Testin has been proposed to have an open and a closed conformation based on the observation that its N-terminal half and C-terminal half directly interact. Here we extend the testin conformational model by demonstrating that testin can also form an antiparallel homodimer. In support of this extended model we determined that the testin region (amino acids 52-233) harbouring the PET domain interacts with the C-terminal LIM1-2 domains in vitro and in cells, and assign a critical role to tyrosine 288 in this interaction

Quantitative Kinetic Study of the Actin-Bundling Protein L-Plastin and of Its Impact on Actin Turn-Over

BACKGROUND: Initially detected in leukocytes and cancer cells derived from solid tissues, L-plastin/fimbrin belongs to a large family of actin crosslinkers and is considered as a marker for many cancers. Phosphorylation of L-plastin on residue Ser5 increases its F-actin binding activity and is required for L-plastin-mediated cell invasion. METHODOLOGY/PRINCIPAL FINDINGS: To study the kinetics of L-plastin and the impact of L-plastin Ser5 phosphorylation on L-plastin dynamics and actin turn-over in live cells, simian Vero cells were transfected with GFP-coupled WT-L-plastin, Ser5 substitution variants (S5/A, S5/E) or actin and analyzed by fluorescence recovery after photobleaching (FRAP). FRAP data were explored by mathematical modeling to estimate steady-state reaction parameters. We demonstrate that in Vero cell focal adhesions L-plastin undergoes rapid cycles of association/dissociation following a two-binding-state model. Phosphorylation of L-plastin increased its association rates by two-fold, whereas dissociation rates were unaffected. Importantly, L-plastin affected actin turn-over by decreasing the actin dissociation rate by four-fold, increasing thereby the amount of F-actin in the focal adhesions, all these effects being promoted by Ser5 phosphorylation. In MCF-7 breast carcinoma cells, phorbol 12-myristate 13-acetate (PMA) treatment induced L-plastin translocation to de novo actin polymerization sites in ruffling membranes and spike-like structures and highly increased its Ser5 phosphorylation. Both inhibition studies and siRNA knock-down of PKC isozymes pointed to the involvement of the novel PKC-delta isozyme in the PMA-elicited signaling pathway leading to L-plastin Ser5 phosphorylation. Furthermore, the L-plastin contribution to actin dynamics regulation was substantiated by its association with a protein complex comprising cortactin, which is known to be involved in this process. CONCLUSIONS/SIGNIFICANCE: Altogether these findings quantitatively demonstrate for the first time that L-plastin contributes to the fine-tuning of actin turn-over, an activity which is regulated by Ser5 phosphorylation promoting its high affinity binding to the cytoskeleton. In carcinoma cells, PKC-delta signaling pathways appear to link L-plastin phosphorylation to actin polymerization and invasion

Variation in Structure and Process of Care in Traumatic Brain Injury: Provider Profiles of European Neurotrauma Centers Participating in the CENTER-TBI Study.

INTRODUCTION: The strength of evidence underpinning care and treatment recommendations in traumatic brain injury (TBI) is low. Comparative effectiveness research (CER) has been proposed as a framework to provide evidence for optimal care for TBI patients. The first step in CER is to map the existing variation. The aim of current study is to quantify variation in general structural and process characteristics among centers participating in the Collaborative European NeuroTrauma Effectiveness Research in Traumatic Brain Injury (CENTER-TBI) study. METHODS: We designed a set of 11 provider profiling questionnaires with 321 questions about various aspects of TBI care, chosen based on literature and expert opinion. After pilot testing, questionnaires were disseminated to 71 centers from 20 countries participating in the CENTER-TBI study. Reliability of questionnaires was estimated by calculating a concordance rate among 5% duplicate questions. RESULTS: All 71 centers completed the questionnaires. Median concordance rate among duplicate questions was 0.85. The majority of centers were academic hospitals (n = 65, 92%), designated as a level I trauma center (n = 48, 68%) and situated in an urban location (n = 70, 99%). The availability of facilities for neuro-trauma care varied across centers; e.g. 40 (57%) had a dedicated neuro-intensive care unit (ICU), 36 (51%) had an in-hospital rehabilitation unit and the organization of the ICU was closed in 64% (n = 45) of the centers. In addition, we found wide variation in processes of care, such as the ICU admission policy and intracranial pressure monitoring policy among centers. CONCLUSION: Even among high-volume, specialized neurotrauma centers there is substantial variation in structures and processes of TBI care. This variation provides an opportunity to study effectiveness of specific aspects of TBI care and to identify best practices with CER approaches

Variation in general supportive and preventive intensive care management of traumatic brain injury: a survey in 66 neurotrauma centers participating in the Collaborative European NeuroTrauma Effectiveness Research in Traumatic Brain Injury (CENTER-TBI) study

Abstract Background General supportive and preventive measures in the intensive care management of traumatic brain injury (TBI) aim to prevent or limit secondary brain injury and optimize recovery. The aim of this survey was to assess and quantify variation in perceptions on intensive care unit (ICU) management of patients with TBI in European neurotrauma centers. Methods We performed a survey as part of the Collaborative European NeuroTrauma Effectiveness Research in Traumatic Brain Injury (CENTER-TBI) study. We analyzed 23 questions focused on: 1) circulatory and respiratory management; 2) fever control; 3) use of corticosteroids; 4) nutrition and glucose management; and 5) seizure prophylaxis and treatment. Results The survey was completed predominantly by intensivists (n = 33, 50%) and neurosurgeons (n = 23, 35%) from 66 centers (97% response rate). The most common cerebral perfusion pressure (CPP) target was > 60 mmHg (n = 39, 60%) and/or an individualized target (n = 25, 38%). To support CPP, crystalloid fluid loading (n = 60, 91%) was generally preferred over albumin (n = 15, 23%), and vasopressors (n = 63, 96%) over inotropes (n = 29, 44%). The most commonly reported target of partial pressure of carbon dioxide in arterial blood (PaCO2) was 36–40 mmHg (4.8–5.3 kPa) in case of controlled intracranial pressure (ICP) < 20 mmHg (n = 45, 69%) and PaCO2 target of 30–35 mmHg (4–4.7 kPa) in case of raised ICP (n = 40, 62%). Almost all respondents indicated to generally treat fever (n = 65, 98%) with paracetamol (n = 61, 92%) and/or external cooling (n = 49, 74%). Conventional glucose management (n = 43, 66%) was preferred over tight glycemic control (n = 18, 28%). More than half of the respondents indicated to aim for full caloric replacement within 7 days (n = 43, 66%) using enteral nutrition (n = 60, 92%). Indications for and duration of seizure prophylaxis varied, and levetiracetam was mostly reported as the agent of choice for both seizure prophylaxis (n = 32, 49%) and treatment (n = 40, 61%). Conclusions Practice preferences vary substantially regarding general supportive and preventive measures in TBI patients at ICUs of European neurotrauma centers. These results provide an opportunity for future comparative effectiveness research, since a more evidence-based uniformity in good practices in general ICU management could have a major impact on TBI outcome

The region 52–233 containing the PET domain of testin directly interacts with LIM1-2 domains in cells.

<p>Immunofluorescence staining of myc/mito-coupled (bait) and GFP-coupled (prey) testin constructs in HeLa cells. Myc signal (red), GFP signal (green) and merge are shown for each condition. Colocalisation is observed for NT-myc/mito and LIM1-3-GFP (A), PET^52-233-myc/mito and LIM1-3-GFP (B) and PET^52-233-myc/mito and LIM1-2-GFP (C). Absence of colocalisation is observed for ΔPET^Δ92-199-myc/mito and LIM1-2-GFP (D). See also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0177879#pone.0177879.s004" target="_blank">S4 Fig</a>.</p

Conformational model of the testin protein.

<p>Testin can adopt an open active monomeric or a closed inactive monomeric conformation (as proposed by Garvalov <i>et al</i>. [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0177879#pone.0177879.ref001" target="_blank">1</a>]) or an antiparallel dimeric conformation (this work). The activity status of the dimer is unknown (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0177879#sec007" target="_blank">Discussion</a>). The interaction between PET^52-233 and the LIM1-2 domains underlies formation of the dimer and/or closed monomer conformation.</p

Full length testin interacts with full length testin in vitro.

<p>A) General Scheme of affinity purification used to produce data in several figures, to demonstrate an interaction of testin with other testin variants. In panel B of this figure, recombinant GST-FL was either used as bait on glutathione-sepharose or in parallel treated with thrombin to remove GST to be used as prey in an untagged form. Thrombin was inactivated prior to addition of this soluble form (input: I) to the resin with GST bound protein. After washing the resin, bound proteins were eluted with heated sample buffer and thus contain both bait and potential prey proteins (affinity purified: P). Proteins were detected either by Western Blotting (Figs 2B–2F and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0177879#pone.0177879.g004" target="_blank">4D</a>) or Coomassie (Figs <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0177879#pone.0177879.g004" target="_blank">4A–4C</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0177879#pone.0177879.g005" target="_blank">5A–5D</a>), T = temperature. B) Immobilised recombinant GST-FL (bait) was incubated with soluble recombinant untagged FL (prey). A Western blot using anti-testin (green) and anti-GST (red) antibodies of the input (I) and proteins on the resin (P) is shown. FL prey (approx. 50 kDa) was present on the resin together with the bait GST-FL (lane ‘GST-FL/P’). Input (I) shows the untagged FL in solution. Recombinant immobilised GST was used as a negative control (lane ‘GST/P’). C) Western blot analysis (anti-testin (green), anti-GST (red)) of a mock buffer control incubated with immobilised GST-FL on resin. Similar as in B, the buffer contains inactivated thrombin but no soluble FL prey. Untagged FL is absent on the resin (P) (compare to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0177879#pone.0177879.g002" target="_blank">Fig 2B</a>, lane ‘GST-FL/P’) indicating that possible residual thrombin activity is not cleaving the GST-FL on the resin. D, E, F) Immobilised recombinant GST-FL (bait) was incubated with soluble untagged Evl (positive control, D) or cofilin (negative control, F) as preys. Immobilised GST was incubated with Evl (prey) and used as additional negative control (E). Western blot analysis of inputs (I) and proteins on the resin (P) is shown using anti testin (green), anti-Evl, anti-cofilin and anti-GST (red) antibodies. Untagged Evl (prey) is present on the GST-FL resin (lane P, 2D). Positions of bait, prey and negative control bait (Neg Ctr) are indicated in each panel. M: marker proteins (kDa).</p

The PET domain of testin is not sufficient for interaction with LIM1-2 domains <i>in vitro</i>.

<p>The experimental setup is similar as the scheme in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0177879#pone.0177879.g002" target="_blank">Fig 2A</a>. Immobilised GST-LIM1-2 on glutathione resin was incubated with untagged PET^52-233(A), PET^92-199(B), PET^52-199 (C) or PET^92-233 (D) in solution, used as preys. Coomassie stained SDS-PAGE analysis is shown: input (I) shows the untagged prey protein prior to incubation with the resin. Lanes indicated with P1 show the proteins present on the resin. We here included an extra negative control: the immobilised baits on the resin were mock-incubated with a solution lacking the soluble preys as in some cases the bait construct is prone to degradation during immobilization on the resin (lanes labelled P2). GST-cofilin resin was used as second negative control in each setup (lanes P3). Untagged prey protein PET^52-233 bound to the GST- LIM1-2 testin variant immobilised on the resin is highlighted by a red box (A). Positions of bait, prey and negative control (Neg Ctr) bait are indicated in each panel, M: marker proteins (kDa).</p