RESUMEN
PtdIns(3,4) P (2), a breakdown product of the lipid second messenger PtdIns(3,4,5) P (3), is a key signalling molecule in pathways controlling various cellular events. Cellular levels of PtdIns(3,4) P (2) are elevated upon agonist stimulation, mediating downstream signalling pathways by recruiting proteins containing specialized lipid-binding modules, such as the pleckstrin homology (PH) domain. A recently identified protein, TAPP1 (tandem-PH-domain-containing protein 1), has been shown to interact in vitro with high affinity and specificity with PtdIns(3,4) P (2) through its C-terminal PH domain. In the present study, we have utilized this PH domain tagged with glutathione S-transferase (GST-TAPP1-PH) as a probe in an on-section immunoelectron microscopy labelling procedure, mapping the subcellular distribution of PtdIns(3,4) P (2). As expected, we found accumulation of PtdIns(3,4) P (2) at the plasma membrane in response to the agonists platelet-derived growth factor and hydrogen peroxide. Importantly, however, we also found agonist stimulated PtdIns(3,4) P (2) labelling of intracellular organelles, including the endoplasmic reticulum and multivesicular endosomes. Expression of the 3-phosphatase PTEN (phosphatase and tensin homologue deleted on chromosome 10) in PTEN-null U87MG cells revealed differential sensitivity of these lipid pools to the enzyme. These data suggest a role for PtdIns(3,4) P (2) in endomembrane function.
Asunto(s)
Proteínas Sanguíneas/genética , Proteínas Portadoras/genética , Péptidos y Proteínas de Señalización Intracelular , Espacio Intracelular/metabolismo , Proteínas de la Membrana/genética , Fosfatos de Fosfatidilinositol/metabolismo , Fosfoproteínas/genética , Homología de Secuencia de Ácido Nucleico , Animales , Línea Celular , Línea Celular Tumoral , Sondas de ADN/genética , Regulación hacia Abajo/efectos de los fármacos , Retículo Endoplásmico/química , Retículo Endoplásmico/efectos de los fármacos , Humanos , Peróxido de Hidrógeno/farmacología , Membranas Intracelulares/química , Membranas Intracelulares/efectos de los fármacos , Espacio Intracelular/química , Ratones , Microscopía Inmunoelectrónica/métodos , Fosfohidrolasa PTEN , Péptidos/genética , Monoéster Fosfórico Hidrolasas/biosíntesis , Factor de Crecimiento Derivado de Plaquetas/farmacología , Estructura Terciaria de Proteína/genética , Coloración y Etiquetado/métodos , Células 3T3 Swiss/química , Células 3T3 Swiss/efectos de los fármacos , Proteínas Supresoras de Tumor/biosíntesisRESUMEN
BACKGROUND: 2,3-butanedione monoxime (BDM) has been widely used as a non-muscle myosin inhibitor to investigate the role of non-muscle myosinII in the process of actin retrograde flow and other actin cytoskeletal processes. Recent reports show that BDM does not inhibit any non-muscle myosins so far tested, including nm-myosinII, prompting the question, how were these process affected in BDM studies? RESULTS: We have found that treatment of mammalian cells with BDM for only 1 min blocks actin incorporation at the leading edge in a permeabilized cell system. We show that inhibition of actin incorporation occurs through de-localization of leading edge proteins involved in actin polymerization--the Arp2/3 complex, WAVE, and VASP--that de-localize concomitantly with the leading edge actin network. CONCLUSION: De-localization of actin leading edge components by BDM treatment is a newly described effect of this compound. It may explain many of the results previously ascribed to inhibition of non-muscle myosinII by BDM, particularly in studies of leading edge dynamics. Though this effect of BDM is intriguing, future studies probing actin dynamics at the leading edge should use more potent and specific inhibitors.