A RAC-GEF network critical for early intestinal tumourigenesis.

RAC1 activity is critical for intestinal homeostasis, and is required for hyperproliferation driven by loss of the tumour suppressor gene Apc in the murine intestine. To avoid the impact of direct targeting upon homeostasis, we reasoned that indirect targeting of RAC1 via RAC-GEFs might be effective. Transcriptional profiling of Apc deficient intestinal tissue identified Vav3 and Tiam1 as key targets. Deletion of these indicated that while TIAM1 deficiency could suppress Apc-driven hyperproliferation, it had no impact upon tumourigenesis, while VAV3 deficiency had no effect. Intriguingly, deletion of either gene resulted in upregulation of Vav2, with subsequent targeting of all three (Vav2-/- Vav3-/- Tiam1-/-), profoundly suppressing hyperproliferation, tumourigenesis and RAC1 activity, without impacting normal homeostasis. Critically, the observed RAC-GEF dependency was negated by oncogenic KRAS mutation. Together, these data demonstrate that while targeting RAC-GEF molecules may have therapeutic impact at early stages, this benefit may be lost in late stage disease.

The ATG16L1 risk allele associated with Crohn's disease results in a Rac1-dependent defect in dendritic cell migration that is corrected by thiopurines.

Thiopurines are commonly used drugs in the therapy of Crohn's disease, but unfortunately only show a 30% response rate. The biological basis for the thiopurine response is unclear, thus hampering patient selection prior to treatment. A genetic risk factor associated specifically with Crohn's disease is a variant in ATG16L1 that reduces autophagy. We have previously shown that autophagy is involved in dendritic cell (DC)-T-cell interactions and cytoskeletal regulation. Here we further investigated the role of autophagy in DC cytoskeletal modulation and cellular trafficking. Autophagy-deficient DC displayed loss of filopodia, altered podosome distribution, and increased membrane ruffling, all consistent with increased cellular adhesion. Consequently, autophagy-deficient DC showed reduced migration. The cytoskeletal aberrations were mediated through hyperactivation of Rac1, a known thiopurine target. Indeed thiopurines restored the migratory defects in autophagy-deficient DC. Clinically, the ATG16L1 risk variant associated with increased response to thiopurine treatment in patients with Crohn's disease but not ulcerative colitis. These results suggest that the association between ATG16L1 and Crohn's disease is mediated at least in part through Rac1 hyperactivation and subsequent defective DC migration. As this phenotype can be corrected using thiopurines, ATG16L1 genotyping may be useful in the identification of patients that will benefit most from thiopurine treatment.

Sequencing of the von Hippel-Lindau gene in canine renal carcinoma.

BACKGROUND: Similarities in human and canine renal cell carcinoma (RCC) epidemiology and biologic behavior suggest that molecular mechanisms of tumorigenesis may be similar in both species. Approximately 75% of RCC in people are of the clear cell subtype, up to 85% of which are associated with mutation of the von Hippel-Lindau (VHL) gene. The canine VHL coding deoxyribonucleic acid (DNA) shares 90% identity with the human VHL gene. OBJECTIVE: To determine whether or not RCC in dogs are associated with VHL mutations, and if so determine the prevalence, type, and location of these mutations. ANIMALS: Thirteen dogs with RCC, 2 dogs with primary renal sarcomas, and 10 dogs without neoplastic kidney disease. METHODS: DNA was extracted from paraffin-embedded RCC tissue; DNA extracts from paraffin-embedded and snap-frozen nonneoplastic canine kidneys and canine whole blood were used as negative controls. Polymerase chain reaction and sequencing of the 3 VHL exons was performed, and results compared with the accessioned canine sequence. RESULTS: All VHL exons were amplified from 9 of 13 canine RCC samples, both renal sarcomas, 8 of 10 nonneoplastic kidney samples, and canine whole blood; only exon 2 could be amplified from 2 RCC samples. Mutations were not identified in any exons. A maximal prevalence of 33.6% for VHL mutations in canine RCC was determined. CONCLUSION AND CLINICAL IMPORTANCE: Although similarities between canine and human RCC merit further investigation of the dog as a model for some subtypes of renal tumors, the lower prevalence of VHL mutations suggests that oncogenesis in these 2 species differs.

Contact dynamics during keratocyte motility.

BACKGROUND: Keratocytes are specialised, rapidly moving cells that generate substantial contractile force perpendicular to their direction of locomotion. Potential roles for contractile force in cell motility include cell-body transport, regulation of adhesion, and retraction of the cell's trailing edge. RESULTS: To investigate contact dynamics, we used simultaneous confocal fluorescence and interference reflection microscopy to image keratocytes injected with fluorescent vinculin. We found that contacts formed behind the leading edge and grew beneath both the lamellipodium and the cell body. Contacts in the middle of the cell remained stationary relative to the substrate and began to disassemble as the cell body passed over them. In contrast, contacts in the lobes of the cell grew continuously and more rapidly, incorporated more vinculin, and slid inwards towards the sides of the cell body. Contact sliding often led to merging of contacts before their removal from the substrate. CONCLUSIONS: We suggest a synthesis of two existing, apparently conflicting models for keratocyte motility, in which network contraction progressively reorients actin filaments using the contacts as pivots, forming bundles that then generate lateral tension by a sliding-filament mechanism. Contact dynamics vary between the middle of the cell and the lobes. We propose that laterally opposed contractile forces first enhance contact growth and stability, but escalating force eventually pulls contacts from the substrate at the back of the cell, without interfering with the cell's forward progress.

Visualising the actin cytoskeleton.

The actin cytoskeleton is a dynamic filamentous network whose formation and remodeling underlies the fundamental processes of cell motility and shape determination. To serve these roles, different compartments of the actin cytoskeleton engage in forming specific coupling sites between neighbouring cells and with the underlying matrix, which themselves serve signal transducing functions. In this review, we focus on methods used to visualise the actin cytoskeleton and its dynamics, embracing the use of proteins tagged with conventional fluorophores and green fluorescent protein. Included also is a comparison of cooled CCD technology, confocal and 2-photon fluorescence microscopy of living and fixed cells, as well as a critique of current procedures for electron microscopy.

Coordination of protrusion and translocation of the keratocyte involves rolling of the cell body.

We have investigated the relationship between lamellipodium protrusion and forward translocation of the cell body in the rapidly moving keratocyte. It is first shown that the trailing, ellipsoidal cell body rotates during translocation. This was indicated by the rotation of the nucleus and the movement of cytoplasmic organelles, as well as of exogenously added beads used as markers. Activated or Con A-coated fluorescent beads that were overrun by cells were commonly endocytosed and rotated with the internal organelles. Alternatively, beads applied to the rear of the cell body via a micropipette adhered to the dorsal cell surface and also moved forward, indicating that both exterior and underlying cortical elements participated in rotation. Manipulation of keratocytes with microneedles demonstrated that pushing or restraining the cell body in the direction of locomotion, and squeezing it against the substrate, which temporarily increased the intracellular pressure, did not effect the rate of lamellipodium protrusion. Rotation and translocation of the cell body continued momentarily after arrest of lamellipodium protrusion by cytochalasin B, indicating that these processes were not directly dependent on actin polymerization. The cell body was commonly flanked by phase-dense "axles," extending from the cell body into the lamellipodium. Phalloidin staining showed these to be comprised of actin bundles that splayed forward into the flanks of the lamellipodium. Disruption of the bundles on one side of the nucleus by traumatic microinjection resulted in rapid retraction of the cell body in the opposite direction, indicating that the cell body was under lateral contractile stress. Myosin II, which colocalizes with the actin bundles, presumably provides the basis of tension generation across and traction of the cell body. We propose that the basis of coupling between lamellipodium protrusion and translocation of the cell body is a flow of actin filaments from the front, where they are nucleated and engage in protrusion, to the rear, where they collaborate with myosin in contraction. Myosin-dependent force is presumably transmitted from the ends of the cell body into the flanks of the lamellipodium via the actin bundles. This force induces the spindle-shaped cell body to roll between the axles that are created continuously from filaments supplied by the advancing lamellipodium.

Calponin reduces shortening velocity in skinned taenia coli smooth muscle fibres.

Calponin (4.1-5.9 microM, pig stomach) inhibited maximal shortening velocity (Vmax) by 20-25% with only minor influence on force in skinned smooth muscle from guinea-pig taenia coli activated at different Ca2+ levels and with thiophosphorylation. Similar results were obtained with a fragment of the N-terminal 1-228 amino acids engineered using a mouse cDNA construct (5.4 microM). Both the native calponin and the fragment inhibited actin filament sliding in a graded manner in an in vitro motility assay. We conclude that calponin influences the kinetics of the actin-myosin interaction in the organised smooth muscle contractile system and that engineered fragments of calponin can be used to probe its action in muscle fibres. The effects can be due to an introduction of an internal load during filament sliding, possibly by decreasing the detachment rates and increasing the cross-bridge time spent in the attached state.

Isolation and characterization of mutants of Propionibacterium strains.

Procedures were developed to isolate and characterize mutants of strains of dairy propionibacteria. These procedures included the construction of minimal defined media to support growth of the strains, optimization of conditions of exposure of the strains to nitrosoguanidine, and identification of the phenotypes of the mutants that were generated. The minimal defined medium contained inorganic salts, adenosine, three vitamins, and sodium lactate as the carbon source, with cysteine, methionine, or cysteine plus methionine added as required by some strains. For mutagenesis, cells were exposed to either 100, 200, or 1000 micrograms/ml nitrosoguanidine, depending on the sensitivity of the strain, for 60 min at 35 degrees C. At least nine stable mutants were isolated and characterized for each of the five strains under study. The most frequent mutations generated were requirements for arginine, histidine, methionine, and uracil and alteration in pigment production.