RESUMEN
Organization and dynamics of focal adhesion proteins have been well characterized in cells grown on two-dimensional (2D) cell culture surfaces. However, much less is known about the dynamic association of these proteins in the 3D microenvironment. Limited imaging technologies capable of measuring protein interactions in real time and space for cells grown in 3D is a major impediment in understanding how proteins function under different environmental cues. In this study, we applied the nano-scale precise imaging by rapid beam oscillation (nSPIRO) technique and combined the scaning-fluorescence correlation spectroscopy (sFCS) and the number and molecular brightness (N&B) methods to investigate paxillin and actin dynamics at focal adhesions in 3D. Both MDA-MB-231 cells and U2OS cells produce elongated protrusions with high intensity regions of paxillin in cell grown in 3D collagen matrices. Using sFCS we found higher percentage of slow diffusing proteins at these focal spots, suggesting assembling/disassembling processes. In addition, the N&B analysis shows paxillin aggregated predominantly at these focal contacts which are next to collagen fibers. At those sites, actin showed slower apparent diffusion rate, which indicated that actin is either polymerizing or binding to the scaffolds in these locals. Our findings demonstrate that by multiplexing these techniques we have the ability to spatially and temporally quantify focal adhesion assembly and disassembly in 3D space and allow the understanding tumor cell invasion in a more complex relevant environment.
Asunto(s)
Línea Celular/ultraestructura , Adhesiones Focales/química , Adhesiones Focales/metabolismo , Espectrometría de Fluorescencia/métodos , Actinas/metabolismo , Técnicas de Cultivo de Célula , Colágeno Tipo I/química , Humanos , Nanotecnología/métodos , Paxillin/metabolismo , Unión Proteica , Conformación ProteicaRESUMEN
Proper assembly of mitotic spindles requires Hice1, a spindle-associated protein. Hice1 possesses direct microtubule binding activity at its N-terminal region and contributes to intraspindle microtubule nucleation as a subunit of the Augmin complex. However, whether microtubule binding activity of Hice1 is modulated by mitotic regulators remains unexplored. Here, we found that Aurora-A kinase, a major mitotic kinase, specifically binds to and phosphorylates Hice1. We identified four serine/threonine clusters on Hice1 that can be phosphorylated by Aurora-A in vitro. Of the four clusters, the Ser/Thr-17-21 cluster was the most critical for bipolar spindle assembly, whereas other phospho-deficient point mutants had a minimal effect on spindle assembly. Immunostaining with a phospho-Ser-19/20 phospho-specific antibody revealed that phosphorylated Hice1 primarily localizes to spindle poles during prophase to metaphase but gradually diminishes after anaphase. Consistently, the phospho-mimic 17-21E mutant reduced microtubule binding activity in vitro and diminished localization to spindles in vivo. Furthermore, expression of the 17-21E mutant led to decreased association of Fam29a, an Augmin component, with spindles. On the other hand, expression of the phospho-deficient 17-21A mutant permitted intraspindle nucleation but delayed the separation of early mitotic spindle poles and the timely mitotic progression. Taken together, these results suggest that Aurora-A modulates the microtubule binding activity of Hice1 in a spatiotemporal manner for proper bipolar spindle assembly.
Asunto(s)
Metafase/fisiología , Proteínas Asociadas a Microtúbulos/metabolismo , Microtúbulos/metabolismo , Profase/fisiología , Proteínas Serina-Treonina Quinasas/metabolismo , Huso Acromático/metabolismo , Aurora Quinasas , Línea Celular Tumoral , Humanos , Proteínas Asociadas a Microtúbulos/genética , Microtúbulos/genética , Mutación , Fosforilación/fisiología , Proteínas Serina-Treonina Quinasas/genética , Estructura Terciaria de Proteína , Huso Acromático/genéticaRESUMEN
RB plays an essential role in DNA damage-induced growth arrest and regulates the expression of several factors essential for DNA repair machinery. However, how RB coordinates DNA damage response through transcriptional regulation of genes involved in growth arrest remains largely unexplored. We examined whether RB can mediate the response to DNA damage through modulation of ZBRK1, a zinc finger-containing transcriptional repressor that can modulate the expression of GADD45A, a DNA damage response gene, to induce cell cycle arrest in response to DNA damage. We found that the ZBRK1 promoter contains an authentic E2F-recognition sequence that specifically binds E2F1, but not E2F4 or E2F6, together with chromatin remodeling proteins CtIP and CtBP to form a repression complex that suppresses ZBRK1 transcription. Furthermore, loss of RB-mediated transcriptional repression led to an increase in ZBRK1 transcript levels, correlating with increased sensitivity to ultraviolet (UV) and methyl methanesulfonate-induced DNA damage. Taken together, these results suggest that the RB·CtIP (CtBP interacting protein)/CtBP (C terminus-binding protein) /E2F1 complex plays a critical role in ZBRK1 transcriptional repression, and loss of this repression may contribute to cellular sensitivity of DNA damage, ultimately leading to carcinogenesis.
Asunto(s)
Daño del ADN/fisiología , Factor de Transcripción E2F1/metabolismo , Complejos Multiproteicos/metabolismo , Proteínas Represoras/biosíntesis , Elementos de Respuesta/fisiología , Proteína de Retinoblastoma/metabolismo , Transcripción Genética/fisiología , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Ciclo Celular/efectos de los fármacos , Ciclo Celular/fisiología , Ciclo Celular/efectos de la radiación , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Línea Celular Tumoral , Ensamble y Desensamble de Cromatina/efectos de los fármacos , Ensamble y Desensamble de Cromatina/fisiología , Ensamble y Desensamble de Cromatina/efectos de la radiación , Daño del ADN/efectos de los fármacos , Daño del ADN/efectos de la radiación , Factor de Transcripción E2F1/genética , Endodesoxirribonucleasas , Humanos , Mesilatos/farmacología , Complejos Multiproteicos/genética , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Proteínas Represoras/genética , Proteína de Retinoblastoma/genética , Transcripción Genética/efectos de los fármacos , Transcripción Genética/genética , Rayos Ultravioleta/efectos adversosRESUMEN
The high mobility group AT-hook 2 (HMGA2), a DNA architectural protein, is highly regulated during development and plays an important role in tumorigenesis. Indeed, HMGA2 was overexpressed in many different kinds of tumors. However, the mechanisms regulating HMGA2 expression remain elusive. Using microarray analysis, we found that HMGA2, along with a dozen of other genes, was co-repressed by ZBRK1, BRCA1, and CtIP. BRCA1 exerts its transcriptional repression activity through interaction with the transcriptional repressor ZBRK1 in the central domain, and with CtIP in the C-terminal BRCT domain. Here, we show that ZBRK1, BRCA1, and CtIP form a repression complex that coordinately regulates HMGA2 expression via a ZBRK1 recognition site in the HMGA2 promoter. Depletion of any of the proteins in this complex via adenoviral RNA interference in MCF10A mammary epithelial cells activates HMGA2 expression, resulting in increased colony formation in soft agar. Similarly, depletion of ZBRK1, or ectopic overexpression of HMGA2, in MCF10A cells induces abnormal acinar size with increased cell number and inhibits normal acinar formation. Consistently, many BRCA1-deficient mouse breast tumors express higher levels of HMGA2 than BRCA1-proficient tumors. These results suggest that activation of HMGA2 gene expression through derepression of the ZBRK1/BRCA1/CtIP complex is a significant step in accelerating breast tumorigenesis.
Asunto(s)
Proteína BRCA1/metabolismo , Neoplasias de la Mama/metabolismo , Proteínas Portadoras/metabolismo , Proteína HMGA2/metabolismo , Proteínas Nucleares/metabolismo , Proteínas Represoras/metabolismo , Animales , Proteína BRCA1/genética , Western Blotting , Neoplasias de la Mama/genética , Proteínas Portadoras/genética , Línea Celular , Línea Celular Tumoral , Inmunoprecipitación de Cromatina , Electroforesis en Gel de Poliacrilamida , Endodesoxirribonucleasas , Proteína HMGA2/genética , Humanos , Neoplasias Mamarias Animales/genética , Neoplasias Mamarias Animales/metabolismo , Ratones , Ratones Noqueados , Proteínas Nucleares/genética , ARN Interferente Pequeño , Proteínas Represoras/genética , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Class switch DNA recombination (CSR) and somatic hypermutation (SHM) are central to the maturation of the Ab response. Both processes involve DNA mismatch repair (MMR). MMR proteins are recruited to dU:dG mispairs generated by activation-induced cytidine deaminase-mediated deamination of dC residues, thereby promoting S-S region synapses and introduction of mismatches (mutations). The MutL homolog Mlh3 is the last complement of the mammalian set of MMR proteins. It is highly conserved in evolution and is essential to meiosis and microsatellite stability. We used the recently generated knockout mlh3(-/-) mice to address the role of Mlh3 in CSR and SHM. We found that Mlh3 deficiency alters both CSR and SHM. mlh3(-/-) B cells switched in vitro to IgG and IgA but displayed preferential targeting of the RGYW/WRCY (R = A or G, Y = C or T, W = A or T) motif by Sgamma1 and Sgamma3 breakpoints and introduced more insertions and fewer donor/acceptor microhomologies in Smu-Sgamma1 and Smu-Sgamma3 DNA junctions, as compared with mlh3(+/+) B cells. mlh3(-/-) mice showed only a slight decrease in the frequency of mutations in the intronic DNA downstream of the rearranged J(H)4 gene. However, the residual mutations were altered in spectrum. They comprised a decreased proportion of mutations at dA/dT and showed preferential RGYW/WRCY targeting by mutations at dC/dG. Thus, the MMR Mlh3 protein plays a role in both CSR and SHM.