RESUMEN
Formins constitute a large family of proteins that regulate the dynamics and organization of both the actin and microtubule cytoskeletons. Previously we showed that the formin mDia1 helps tether microtubules at the cell cortex, acting downstream of the ErbB2 receptor tyrosine kinase. Here we further study the contributions of mDia1 and its two most closely related formins, mDia2 and mDia3, to cortical microtubule capture and ErbB2-dependent breast carcinoma cell migration. We find that depletion of each of these three formins strongly disrupts chemotaxis without significantly affecting actin-based structures. Further, all three formins are required for formation of cortical microtubules in a nonredundant manner, and formin proteins defective in actin polymerization remain active for microtubule capture. Using affinity purification and mass spectrometry analysis, we identify differential binding partners of the formin-homology domain 2 (FH2) of mDia1, mDia2, and mDia3, which may explain their nonredundant roles in microtubule capture. The FH2 domain of mDia1 specifically interacts with Rab6-interacting protein 2 (Rab6IP2). Further, mDia1 is required for cortical localization of Rab6IP2, and concomitant depletion of Rab6IP2 and IQGAP1 severely disrupts cortical capture of microtubules, demonstrating the coinvolvement of mDia1, IQGAP1, and Rab6IP2 in microtubule tethering at the leading edge.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/fisiología , Proteínas Portadoras/fisiología , Movimiento Celular , Microtúbulos/metabolismo , Actinas/metabolismo , Proteínas Adaptadoras Transductoras de Señales/química , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Animales , Proteínas Portadoras/química , Proteínas Portadoras/metabolismo , Línea Celular Tumoral , Quimiotaxis , Forminas , Humanos , Péptidos y Proteínas de Señalización Intracelular , Proteínas de Microfilamentos/metabolismo , Proteínas Asociadas a Microtúbulos/metabolismo , Proteínas de Hierro no Heme/metabolismo , Estructura Terciaria de Proteína , Conejos , Receptor ErbB-2/metabolismo , Transducción de SeñalRESUMEN
The acquired gain-of-function V617F mutation in the Janus Kinase 2 (JAK2(V617F)) is the main mutation involved in BCR/ABL-negative myeloproliferative neoplasms (MPNs), but its effect on hematopoietic stem cells as a driver of disease emergence has been questioned. Therefore, we reinvestigated the role of endogenous expression of JAK2(V617F) on early steps of hematopoiesis as well as the effect of interferon-α (IFNα), which may target the JAK2(V617F) clone in humans by using knock-in mice with conditional expression of JAK2(V617F) in hematopoietic cells. These mice develop a MPN mimicking polycythemia vera with large amplification of myeloid mature and precursor cells, displaying erythroid endogenous growth and progressing to myelofibrosis. Interestingly, early hematopoietic compartments [Lin-, LSK, and SLAM (LSK/CD48-/CD150+)] increased with the age. Competitive repopulation assays demonstrated disease appearance and progressive overgrowth of myeloid, Lin-, LSK, and SLAM cells, but not lymphocytes, from a low number of engrafted JAK2(V617F) SLAM cells. Finally, IFNα treatment prevented disease development by specifically inhibiting JAK2(V617F) cells at an early stage of differentiation and eradicating disease-initiating cells. This study shows that JAK2(V617F) in mice amplifies not only late but also early hematopoietic cells, giving them a proliferative advantage through high cell cycling and low apoptosis that may sustain MPN emergence but is lost upon IFNα treatment.
Asunto(s)
Apoptosis , Células Madre Hematopoyéticas/citología , Interferón-alfa/metabolismo , Janus Quinasa 2/genética , Janus Quinasa 2/metabolismo , Mutación , Animales , Células de la Médula Ósea/citología , Trasplante de Médula Ósea , Ciclo Celular , Cruzamientos Genéticos , Modelos Animales de Enfermedad , Humanos , Linfocitos/citología , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Trastornos Mieloproliferativos/genética , Fenotipo , Policitemia Vera/genética , Mielofibrosis Primaria/metabolismo , Células Madre/citologíaRESUMEN
The Jak2(V617F) mutation is found in most classical BCR/ABL-negative myeloproliferative neoplasms (MPNs). Usually, heterozygosity of the mutation is associated with essential thrombocythemia (ET) and homozygosity with polycythemia vera (PV). Retrovirally transduced or transgenic animal models have shown that the mutation is sufficient for MPN development but that the level of expression is crucial for MPN phenotypes. Therefore we investigated the effect of an endogenous heterozygous expression of Jak2(V617F) in knock-in (KI) mice. These animals displayed constitutive JAK2 activation and autonomous erythroid progenitor cell growth. Mice suffered from marked polycythemia, granulocytosis and thrombocytosis. Spleens and marrows displayed myeloid trilineage hyperplasia. Most animals survived to develop advanced fibrosis in these organs at around 9 months of age. In conclusion, constitutive heterozygous expression of JAK2(V617F) in mice is not embryo-lethal but results in severe PV-like disease with secondary myelofibrosis and not in ET-like disease as expected from patient study.