XMAP215 promotes microtubule catastrophe by disrupting the growing microtubule end.

The GTP-tubulin cap is widely accepted to protect microtubules against catastrophe. The GTP-cap size is thought to increase with the microtubule growth rate, presumably endowing fast-growing microtubules with enhanced stability. It is unknown what GTP-cap properties permit frequent microtubule catastrophe despite fast growth. Here, we investigate microtubules growing in the presence and absence of the polymerase XMAP215. Using EB1 as a GTP-cap marker, we find that GTP-cap size increases regardless of whether growth acceleration is achieved by increasing tubulin concentration or by XMAP215. Despite increased mean GTP-cap size, microtubules grown with XMAP215 display increased catastrophe frequency, in contrast to microtubules grown with more tubulin, for which catastrophe is abolished. However, microtubules polymerized with XMAP215 have large fluctuations in growth rate; display tapered and curled ends; and undergo catastrophe at faster growth rates and with higher EB1 end-localization. Our results suggest that structural perturbations induced by XMAP215 override the protective effects of the GTP-cap, ultimately driving microtubule catastrophe.

Collective effects of XMAP215, EB1, CLASP2, and MCAK lead to robust microtubule treadmilling.

Microtubule network remodeling is essential for fundamental cellular processes including cell division, differentiation, and motility. Microtubules are active biological polymers whose ends stochastically and independently switch between phases of growth and shrinkage. Microtubule treadmilling, in which the microtubule plus end grows while the minus end shrinks, is observed in cells; however, the underlying mechanisms are not known. Here, we use a combination of computational and in vitro reconstitution approaches to determine the conditions leading to robust microtubule treadmilling. We find that microtubules polymerized from tubulin alone can treadmill, albeit with opposite directionality and order-of-magnitude slower rates than observed in cells. We then employ computational simulations to predict that the combinatory effects of four microtubule-associated proteins (MAPs), namely EB1, XMAP215, CLASP2, and MCAK, can promote fast and sustained plus-end-leading treadmilling. Finally, we experimentally confirm the predictions of our computational model using a multi-MAP, in vitro microtubule dynamics assay to reconstitute robust plus-end-leading treadmilling, consistent with observations in cells. Our results demonstrate how microtubule dynamics can be modulated to achieve a dynamic balance between assembly and disassembly at opposite polymer ends, resulting in treadmilling over long periods of time. Overall, we show how the collective effects of multiple components give rise to complex microtubule behavior that may be used for global network remodeling in cells.

A comprehensive evaluation of Hippo pathway silencing in sarcomas.

TAZ and YAP are transcriptional coactivators negatively regulated by the Hippo pathway that have emerged as key oncoproteins in several cancers including sarcomas. We hypothesized that loss of expression of the Hippo kinases might be a mechanism of activating TAZ and YAP. By immunohistochemistry, TAZ/YAP activated clinical sarcoma samples demonstrated loss of MST1 (47%), MST2 (26%), LATS1 (19%), and LATS2 (27%). Western blot similarly demonstrated loss of MST1 (58%), MST2 (25%), and LATS2 (17%). Treatment with MG132 demonstrated an accumulation of MST2 in 25% of sarcoma cell lines, indicating that proteosomal degradation regulates MST2 expression. qRT-PCR in sarcoma cell lines demonstrated loss of expression of the Hippo kinases at the RNA level, most pronounced in MST1 (42%) and MST2 (25%). 5-azacytidine treatment in sarcoma cell lines modestly reversed expression of predominantly MST1 (8%) and MST2 (17%), indicating CpG island hypermethylation can silence expression of MST1 and MST2. Trichostatin A treatment reversed expression of MST1 (58%) and MST2 (67%), indicating histone deacetylation also plays a role in silencing expression of MST1 and MST2. Loss of expression of the Hippo kinases is frequent in sarcomas and is due to a variety of mechanisms including regulation at the post-translational level and epigenetic silencing.

TAZ and YAP are frequently activated oncoproteins in sarcomas.

TAZ (WWTR1) and YAP are transcriptional coactivators and oncoproteins inhibited by the Hippo pathway. Herein we evaluate 159 sarcomas representing the most prevalent sarcoma types by immunohistochemistry for expression and activation (nuclear localization) of TAZ and YAP. We show that 50% of sarcomas demonstrate activation of YAP while 66% of sarcomas demonstrate activated TAZ. Differential activation of TAZ and YAP are identified in various sarcoma types. At an RNA level, expression of WWTR1 or YAP1 predicts overall survival in undifferentiated pleomorphic sarcoma and dedifferentiated liposarcoma. Immunohistochemistry demonstrates that TAZ and YAP expression and activation are positively correlated with grade in the well-differentiated liposarcoma to dedifferentiated liposarcoma tumor progression sequence as well as conventional chondrosarcomas. TAZ and YAP are constitutively activated oncoproteins in sarcoma cell lines. Knock-down of TAZ and YAP demonstrate differential activity for the two proteins. Verteporfin decreases colony formation in soft agar as well as CTGF expression in sarcoma cell lines harboring activated TAZ and YAP.

Poor Agreement on Classification and Treatment of Subscapularis Tendon Tears.

PURPOSE: To assess the inter- and intraobserver agreement for classification and management of subscapularis tendon pathology based on arthroscopy and magnetic resonance imaging (MRI). METHODS: Twenty-two orthopaedic surgeons from the Multicenter Orthopaedic Outcomes Network (MOON) shoulder group reviewed still arthroscopic and MRI images of the subscapularis tendon from patients with a random assortment of subscapularis morphology. The surgeons were asked to classify the pathology based on 2 systems (Lafosse and Lyons) and choose whether they would repair the tendon and, if so, the method of repair (open or arthroscopic). The survey was administered 3 times to each surgeon. Inter- and intraobserver reliability between testing rounds was determined by kappa analysis. RESULTS: Interobserver reliability on classification of tears was poor based on MRI (k = 0.18 to 0.19) and fair based on arthroscopy (k = 0.26 to 0.29). Interobserver agreement on whether surgical treatment was indicated was fair for both MRI (k = 0.28) and arthroscopy (k = 0.38), while the agreement for type of surgery was poor based on MRI (k = 0.18) and fair based on arthroscopy (k = 0.28). Interobserver agreement did not improve when both MRI and arthroscopy were provided simultaneously (k = 0.24 to 0.30). Intraobserver reliability for classification and treatment was fair to moderate for both MRI (k = 0.32 to 0.50) and arthroscopic imaging (k = 0.39 to 0.56). When considering just those patients with normal tendons, surgeon agreement improved. For all questions, the arthroscopic images had a higher level of agreement among surgeons than the MRI (P < .001). CONCLUSIONS: Although surgeons tended to have higher reliability when presented with arthroscopic images compared with MRI, there was very little agreement on the classification and management of subscapularis tendon tears.

Outcomes after shoulder replacement: comparison between reverse and anatomic total shoulder arthroplasty.

BACKGROUND: Anatomic total shoulder arthroplasty (TSA) and reverse total shoulder arthroplasty (RTSA) are increasingly common procedures employed to treat arthritic conditions. Although TSA is a widely accepted procedure for glenohumeral arthritis with intact rotator cuff, concerns about RTSA persist because of variable complication rates and outcomes. METHODS: This is a prospective, case-control study comparing outcomes and complications after TSA and RTSA. The study included 47 patients undergoing TSA for glenohumeral arthritis and 53 patients undergoing RTSA for rotator cuff tear arthropathy. Average clinical follow-up was more than 2 years in both groups. Major complications included infection, periprosthetic fracture, instability, glenoid loosening, and need for revision surgery. Patient outcome measures included the American Shoulder and Elbow Surgeons score, pain visual analog scale score, and goniometer-measured range of motion. Plain radiographs were reviewed to assess for degree of glenoid lucency in TSA and scapular notching in RTSA. RESULTS: At 2 years, there were no differences in rate of major complications (TSA, 15%; RTSA, 13%; P = .808) or revision surgeries (TSA, 11%; RTSA, 9%). Outcomes assessed by the American Shoulder and Elbow Surgeons score and visual analog scale were also similar between the 2 groups. TSA patients had greater external rotation than RTSA patients did (53° vs 38°; P = .001). Otherwise, forward flexion, abduction, and internal rotation were comparable in range of motion. CONCLUSIONS: TSA and RTSA have similar complication rates, need for revision, patient-reported outcomes, and range of motion at 2 years of follow-up. The use of side-by-side cohorts in this study allows standardized comparison between these 2 shoulder arthroplasty procedures.

Does fungal endophyte infection improve tall fescue's growth response to fire and water limitation?

Invasive species may owe some of their success in competing and co-existing with native species to microbial symbioses they are capable of forming. Tall fescue is a cool-season, non-native, invasive grass capable of co-existing with native warm-season grasses in North American grasslands that frequently experience fire, drought, and cold winters, conditions to which the native species should be better-adapted than tall fescue. We hypothesized that tall fescue's ability to form a symbiosis with Neotyphodium coenophialum, an aboveground fungal endophyte, may enhance its environmental stress tolerance and persistence in these environments. We used a greenhouse experiment to examine the effects of endophyte infection (E+ vs. E-), prescribed fire (1 burn vs. 2 burn vs. unburned control), and watering regime (dry vs. wet) on tall fescue growth. We assessed treatment effects for growth rates and the following response variables: total tiller length, number of tillers recruited during the experiment, number of reproductive tillers, tiller biomass, root biomass, and total biomass. Water regime significantly affected all response variables, with less growth and lower growth rates observed under the dry water regime compared to the wet. The burn treatments significantly affected total tiller length, number of reproductive tillers, total tiller biomass, and total biomass, but treatment differences were not consistent across parameters. Overall, fire seemed to enhance growth. Endophyte status significantly affected total tiller length and tiller biomass, but the effect was opposite what we predicted (E->E+). The results from our experiment indicated that tall fescue was relatively tolerant of fire, even when combined with dry conditions, and that the fungal endophyte symbiosis was not important in governing this ecological ability. The persistence of tall fescue in native grassland ecosystems may be linked to other endophyte-conferred abilities not measured here (e.g., herbivory release) or may not be related to this plant-microbial symbiosis.