Centriolar CPAP/SAS-4 Imparts Slow Processive Microtubule Growth.

Centrioles are fundamental and evolutionarily conserved microtubule-based organelles whose assembly is characterized by microtubule growth rates that are orders of magnitude slower than those of cytoplasmic microtubules. Several centriolar proteins can interact with tubulin or microtubules, but how they ensure the exceptionally slow growth of centriolar microtubules has remained mysterious. Here, we bring together crystallographic, biophysical, and reconstitution assays to demonstrate that the human centriolar protein CPAP (SAS-4 in worms and flies) binds and "caps" microtubule plus ends by associating with a site of ß-tubulin engaged in longitudinal tubulin-tubulin interactions. Strikingly, we uncover that CPAP activity dampens microtubule growth and stabilizes microtubules by inhibiting catastrophes and promoting rescues. We further establish that the capping function of CPAP is important to limit growth of centriolar microtubules in cells. Our results suggest that CPAP acts as a molecular lid that ensures slow assembly of centriolar microtubules and, thereby, contributes to organelle length control.

SAS-6 engineering reveals interdependence between cartwheel and microtubules in determining centriole architecture.

Centrioles are critical for the formation of centrosomes, cilia and flagella in eukaryotes. They are thought to assemble around a nine-fold symmetric cartwheel structure established by SAS-6 proteins. Here, we have engineered Chlamydomonas reinhardtii SAS-6-based oligomers with symmetries ranging from five- to ten-fold. Expression of a SAS-6 mutant that forms six-fold symmetric cartwheel structures in vitro resulted in cartwheels and centrioles with eight- or nine-fold symmetries in vivo. In combination with Bld10 mutants that weaken cartwheel-microtubule interactions, this SAS-6 mutant produced six- to eight-fold symmetric cartwheels. Concurrently, the microtubule wall maintained eight- and nine-fold symmetries. Expressing SAS-6 with analogous mutations in human cells resulted in nine-fold symmetric centrioles that exhibited impaired length and organization. Together, our data suggest that the self-assembly properties of SAS-6 instruct cartwheel symmetry, and lead us to propose a model in which the cartwheel and the microtubule wall assemble in an interdependent manner to establish the native architecture of centrioles.

Coronin 1 regulates cognition and behavior through modulation of cAMP/protein kinase A signaling.

Cognitive and behavioral disorders are thought to be a result of neuronal dysfunction, but the underlying molecular defects remain largely unknown. An important signaling pathway involved in the regulation of neuronal function is the cyclic AMP/Protein kinase A pathway. We here show an essential role for coronin 1, which is encoded in a genomic region associated with neurobehavioral dysfunction, in the modulation of cyclic AMP/PKA signaling. We found that coronin 1 is specifically expressed in excitatory but not inhibitory neurons and that coronin 1 deficiency results in loss of excitatory synapses and severe neurobehavioral disabilities, including reduced anxiety, social deficits, increased aggression, and learning defects. Electrophysiological analysis of excitatory synaptic transmission in amygdala revealed that coronin 1 was essential for cyclic-AMP-protein kinase A-dependent presynaptic plasticity. We further show that upon cell surface stimulation, coronin 1 interacted with the G protein subtype Gαs to stimulate the cAMP/PKA pathway. The absence of coronin 1 or expression of coronin 1 mutants unable to interact with Gαs resulted in a marked reduction in cAMP signaling. Strikingly, synaptic plasticity and behavioral defects of coronin 1-deficient mice were restored by in vivo infusion of a membrane-permeable cAMP analogue. Together these results identify coronin 1 as being important for cognition and behavior through its activity in promoting cAMP/PKA-dependent synaptic plasticity and may open novel avenues for the dissection of signal transduction pathways involved in neurobehavioral processes.

Synthesis and evaluation of biphenyl compounds as kinesin spindle protein inhibitors.

Kinesin spindle protein (KSP), an ATP-dependent motor protein, plays an essential role in bipolar spindle formation during the mitotic phase (M phase) of the normal cell cycle. KSP has emerged as a novel target for antimitotic anticancer drug development. In this work, we synthesized a range of new biphenyl compounds and investigated their properties in vitro as potential antimitotic agents targeting KSP expression. Antiproliferation (MTT (=3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide)) assays, combined with fluorescence-assisted cell sorting (FACS) and Western blot studies analyzing cell-cycle arrest confirmed the mechanism and potency of these biphenyl compounds in a range of human cancer cell lines. Structural variants revealed that functionalization of biphenyl compounds with bulky aliphatic or aromatic groups led to a loss of activity. However, replacement of the urea group with a thiourea led to an increase in antiproliferative activity in selected cell lines. Further studies using confocal fluorescence microscopy confirmed that the most potent biphenyl derivative identified thus far, compound 7, exerts its pharmacologic effect specifically in the M phase and induces monoaster formation. These studies confirm that chemical scope remains for improving the potency and treatment efficacy of antimitotic KSP inhibition in this class of biphenyl compounds.

Outcome, general, and symptom-specific quality of life after various types of parotid resection.

OBJECTIVES/HYPOTHESIS: To document the outcome and impact on general and symptom-specific quality of life (QOL) after various types of parotid resection. STUDY DESIGN: General and symptom-specific QOL assessment at least 1 year after performed surgery. Retrospective data and outcome analysis of patients. METHODS: Between 2004 and 2010, 353 parotid resections in 337 patients were conducted at the Department of Otorhinolaryngology, University Teaching Hospital, St. Mary's Hospital Gelsenkirchen, Gelsenkirchen, Germany. A total of 196 patients fit the inclusion criteria and were available for postoperative evaluation. The general QOL assessment was based on both the global health status and global QOL scales of the European Organisation for Research and Treatment of Cancer (EORTC) Quality-of-Life Questionnaire in 34 patients. Symptom-specific QOL was assessed with the Parotidectomy Outcome Inventory-8 (POI-8). In addition, aesthetic outcome was evaluated with an ordinal scale. RESULTS: Outcome of parotidectomies in benign disease has little impact on general QOL and global health status. However, hypoesthesia or dysesthesia, Frey's syndrome, and cosmetic discontent are quite common and may affect symptom-specific and general QOL. Correlation with extent of surgery and statistically significant differences of patient evaluation for aesthetic outcome, sensory impairment, and Frey's syndrome between various types of limited parotid surgery (enucleation, extracapsular dissection, partial superficial parotidectomy) and superficial parotidectomy could be shown. CONCLUSIONS: An adequate parotid resection technique must be chosen to achieve the least disturbing outcome. In addition, in our patient collective, there was no increased recurrence rate found after limited parotid resection for pleomorphic adenoma or cystadenolymphoma.

Interaction of mammalian end binding proteins with CAP-Gly domains of CLIP-170 and p150(glued).

End binding proteins (EBs) track growing microtubule ends and play a master role in organizing dynamic protein networks. Mammalian cells express up to three different EBs (EB1, EB2, and EB3). Besides forming homodimers, EB1 and EB3 also assemble into heterodimers. One group of EB-binding partners encompasses proteins that harbor CAP-Gly domains. The binding properties of the different EBs towards CAP-Gly proteins have not been systematically investigated. This information is, however, important to compare and contrast functional differences. Here we analyzed the interactions between CLIP-170 and p150(glued) CAP-Gly domains with the three EB homodimers and the EB1-EB3 heterodimer. Using isothermal titration calorimetry we observed that some EBs bind to the individual CAP-Gly domains with similar affinities while others interact with their targets with pronounced differences. We further found that the two types of CAP-Gly domains use alternative mechanisms to target the C-terminal domains of EBs. We succeeded to solve the crystal structure of a complex composed of a heterodimer of EB1 and EB3 C-termini together with the CAP-Gly domain of p150(glued). Together, our results provide mechanistic insights into the interaction properties of EBs and offer a molecular framework for the systematic investigation of their functional differences in cells.

Expression analysis of proline rich 15 (Prr15) in mouse and human gastrointestinal tumors.

Proline rich 15 (Prr15), which encodes a protein of unknown function, is expressed almost exclusively in postmitotic cells both during fetal development and in adult tissues, such as the intestinal epithelium and the testis. To determine if this specific expression is lost in intestinal neoplasias, we examined Prr15 expression by in situ hybridization (ISH) on mouse intestinal tumors caused by different gene mutations, and on human colorectal cancer (CRC) samples. Prr15/PRR15 expression was consistently observed in mouse gastrointestinal (GI) tumors caused by mutations in the Apc gene, as well as in several advanced stage human CRCs. In contrast, no Prr15 expression was detected in intestinal tumors derived from mice carrying mutations in the Smad3, Smad4, or Cdkn1b genes. These findings, combined with the fact that a majority of sporadic human CRCs carry APC mutations, strongly suggest that the expression of Prr15/PRR15 in mouse and human GI tumors is linked, directly or indirectly, to the absence of the APC protein or, more generally, to the disruption of the Wnt signaling pathway.

Orf virus VEGF-E NZ2 promotes paracellular NRP-1/VEGFR-2 coreceptor assembly via the peptide RPPR.

Vascular endothelial growth factors (VEGFs) interact with the receptor tyrosine kinases (RTKs) VEGFR-1, -2, and -3; neuropilins (NRPs); and heparan sulfate (HS) proteoglycans. VEGF RTKs signal to downstream targets upon ligand-induced tyrosine phosphorylation, while NRPs and HS act as coreceptors that lack enzymatic activity yet modulate signal output by VEGF RTKs. VEGFs exist in various isoforms with distinct receptor specificity and biological activity. Here, a series of mammalian VEGF-A splice variants and orf virus VEGF-Es, as well as chimeric and mutant VEGF variants, were characterized to determine the motifs required for binding to NRP-1 in the absence (VEGF-E) or presence (VEGF-A(165)) of an HS-binding sequence. We identified the carboxyterminal peptides RPPR and DKPRR as the NRP-1 binding motifs of VEGF-E and VEGF-A, respectively. RPPR had significantly higher affinity for NRP-1 than DKPRR. VEGFs containing an RPPR motif promoted HS-independent coreceptor complex assembly between VEGFR-2 and NRP-1, independent of whether these receptors were expressed on the same or separate cells grown in cocultures. Functional studies showed that stable coreceptor assembly by VEGF correlated with its ability to promote vessel formation in an embryoid body angiogenesis assay.

Structure-function relationship of CAP-Gly domains.

In all eukaryotes, CAP-Gly proteins control important cellular processes. The molecular mechanisms underlying the functions of CAP-Gly domains, however, are still poorly understood. Here we use the complex formed between the CAP-Gly domain of p150(glued) and the C-terminal zinc knuckle of CLIP170 as a model system to explore the structure-function relationship of CAP-Gly-mediated protein interactions. We demonstrate that the conserved GKNDG motif of CAP-Gly domains is responsible for targeting to the C-terminal EEY/F sequence motifs of CLIP170, EB proteins and microtubules. The CAP-Gly-EEY/F interaction is essential for the recruitment of the dynactin complex by CLIP170 and for activation of CLIP170. Our findings define the molecular basis of CAP-Gly domain function, including the tubulin detyrosination-tyrosination cycle. They further establish fundamental roles for the interaction between CAP-Gly proteins and C-terminal EEY/F sequence motifs in regulating complex and dynamic cellular processes.

Targeting human cancer cells with VEGF receptor-2-directed liposomes.

Antibodies are among the most versatile tools used today to characterize and target molecules in cells and in biological tissues. The development of phage display libraries encoding a large repertoire of single chain antibodies, scFv, allows the rapid and efficient isolation of antibodies specific for almost any type of molecule. A great advantage of such recombinant antibodies is the possibility to functionalize them by introducing new amino acid sequences. This leads to new features that would be difficult to introduce into naturally occurring antibody molecules. This approach has been successfully applied to create molecules with new biological activities, e.g. by generating chimeric scFv antibodies carrying sequences derived from other biomolecules such as blood clotting factors or enzymes. Here, we describe a new antibody isolated from an M13 phage library that recognizes vascular endothelial growth factor receptor 2, VEGFR-2. This antibody, scFvVR-2H9 was coupled to liposomes and used to specifically target VEGFR-2-expressing human cancer cells in culture.

Activation of stress-responsive promoters by ionizing radiation for deployment in targeted gene therapy.

Radiotherapy is one of the principal modalities of cancer treatment, but the delivery of a curative dose of ionizing radiation (IR) to the tumour is frequently limited by the need to protect the normal tissues within the irradiated area from radiation damage. This problem could be circumvented if tumour cells could be selectively sensitized to killing by IR. One way to achieve this goal would be to transduce the tumour cells with expression vectors carrying toxin genes under the control of promoters that are inactive unless induced by IR. For this approach to be successful, two parameters must be met: (i) the expression vector has to be delivered to the tumour or its immediate vicinity (e.g. its vasculature) and (ii) the promoter driving the expression of the toxin gene has to have negligible basal activity, yet has to be activated by clinically-achievable doses of IR. Several vectors that fulfil these criteria are currently reaching clinical trials. In this review, we examine the response of mammalian cells to IR, and the current status of radiation-induced suicide gene therapy that is dependent on this response.

HIV TAT basic peptide is not a high-affinity ligand for VEGF receptor 2.

The 'transactivator of transcription' (TAT) protein of human immunodeficiency virus transforms cells in culture and promotes the development of tumors, so-called Kaposi's sarcoma, in AIDS patients. TAT induces growth and differentiation of blood vessels and has been suggested to directly activate VEGF receptor 2 expressed on endothelial cells through a peptide sequence located between amino acids 46 and 64, the so-called basic domain. This peptide mimics many aspects of TAT function when added to endothelial cells, even when expressed in the context of recombinant chimeric proteins. To define the exact sites of interaction between this peptide and VEGF receptor 2 we performed binding studies with recombinant proteins derived from the extracellular ligand binding domain of VEGF receptor 2. These in vitro binding studies showed that the TAT peptide binds with only low specificity to Ig-like domain 3 of the receptor, while VEGF interacts with receptor-derived proteins encompassing at least extracellular domains 1 through 3. The original concept that the angiogenic properties of TAT basic peptide result from specific, high-affinity interaction with VEGF receptor 2 must therefore be revised. Apparently this peptide interacts with cells in multiple ways: by directly activating acidic cell surface-exposed receptors, by releasing extracellular matrix-bound growth factors such as bFGF and VEGF which then bind to their cognate receptors, and by activating intracellular signalling molecules with which basic peptide interacts upon translocation into cells.