Centrin, a 20 kDa calcium-binding protein, is a constituent of contractile basal body-associated fibers in protists and of various centrosomal structures. A construct inducing centrin RNAi was used to study the effect of centrin deficiency in Chlamydomonas. Transformants contained variable amounts of residual centrin (down to 5% of wildtype) and lacked centrin fibers. They displayed a variable flagellar number phenotype with mostly nonflagellate cells, suggesting that centrin is required for basal body assembly. Furthermore, basal bodies often failed to dock to the plasma membrane and to assemble flagella, and displayed defects in the flagellar root system indicating that centrin deficiency interferes with basal body development. Multiple basal bodies caused the formation of additional microtubular asters, whereas the microtubular cytoskeleton was disordered in most cells without basal bodies. The number of multinucleated cells was increased, indicating that aberrant numbers of basal bodies interfered with the cytokinesis of Chlamydomonas. In contrast to wildtype cells, basal bodies in centrin-RNAi cells were separated from the spindle poles, suggesting a role of centrin in tethering basal bodies to the spindle. To test whether an association with the spindle poles is required for correct basal body segregation, we disrupted centrin fibers in wild-type cells by over-expressing a nonfunctional centrin-GFP. In these cells, basal bodies were disconnected from the spindle but segregation errors were not observed. We propose that basal body segregation in Chlamydomonas depends on an extranuclear array of microtubules independent of the mitotic spindle.

Means to prevent thrombus extension and local recurrence remain suboptimal, in part because of the limited effectiveness of existing thrombolytics. In theory, plasminogen activators could be used for this purpose if they could be anchored to the vascular lumen by targeting stably expressed, noninternalized determinants such as platelet-endothelial-cell adhesion molecule 1 (PECAM-1). We designed a recombinant molecule fusing lowmolecular-weight single-chain prourokinase plasminogen activator (lmw-scuPA) with a single-chain variable fragment (scFv) of a PECAM-1 antibody to generate the prodrug scFv/lmw-scuPA. Cleavage by plasmin generated fibrinolytically active 2-chain lmw-uPA. This fusion protein (1) bound specifically to PECAM-1-expressing cells; (2) was rapidly cleared from blood after intravenous injection; (3) accumulated in the lungs of wild-type C57BL6/J, but not PECAM-1 null mice; and (4) lysed pulmonary emboli formed subsequently more effectively than lmw-scuPA, thereby providing support for the concept of thromboprophylaxis using recombinant scFv-fibrinolytic fusion proteins that target endothelium. ( IntroductionPlasminogen activators (PAs; eg, uPA, urokinase plasminogen activator) help to restore perfusion after thrombotic vascular occlusion, the leading cause of human morbidity and mortality. [1][2][3] However, the clinical utility of PAs is limited by (1) inadequate delivery because of rapid elimination and inactivation en route and ineffective penetration into formed clots; (2) side effects, including extravasation leading to collateral damage in the central nervous system and other tissues; (3) lysis of "physiologic" (hemostatic) clots leading to hemorrhage; and, (4) reperfusion injury following a delay in restoring perfusion, where morbidity correlates with the duration of ischemia. [4][5][6] Clinical settings characterized by a high propensity for thrombosis have been identified, and means to diagnose early clot formation have been developed. 1,2 Although the indications for prophylaxis are known, PAs are not used prophylactically because of their unfavorable pharmacokinetics and side effects. Gene therapy approaches, effective in cell-culture and animal experiments, 7,8 are not practical when the need to enhance fibrinolysis is acute and of short duration. 9 Conceivably, prophylactic delivery of a PA derivative that rapidly restricts and sustains its activity in the vascular lumen can help to lyse nascent clots expeditiously, inhibit propagation of mural thrombi, and reduce the duration of ischemia.For example, PAs can be used for thromboprophylaxis by coupling to carrier red blood cells (RBCs), prolonging circulation and limiting extravasation. 10 This approach may have utility in settings in which RBC transfusion is part of current management. Drug targeting to suitable endothelial-cell-surface determinants 11-13 may provide an alternative approach and, in theory, localize PA activity in the affected intravascular compartment.For example, drugs coupled with antibodies to plateletendothelial ...

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Recombinant tissue factor-based fusion proteins directed against an intraluminal tumor endothelial cell marker induce tumor-selective intravascular coagulation, tumor tissue necrosis, and tumor growth delay.