Borges, Filipe; Gardner, Rui; Lopes, Telma; Calarco, Joseph P; Boavida, Leonor C; Slotkin, R; Martienssen, Robert A; Becker, Jörg D
The male germline in flowering plants differentiates by asymmetric division of haploid uninucleated
microspores, giving rise to a vegetative cell enclosing a smaller generative cell, which eventually undergoes a
second mitosis to originate two sperm cells. The vegetative cell and the sperm cells activate distinct genetic and epigenetic mechanisms to control pollen tube growth and germ cell specification, respectively. Therefore, a comprehensive characterization of these processes relies on efficient methods to isolate each of the different cell types throughout male gametogenesis.
We developed stable transgenic Arabidopsis lines and reliable purification tools based on
Fluorescence-Activated Cell Sorting (FACS) in order to isolate highly pure and...
Gomes-Santos, Carina S S; Itoe, Maurice A; Afonso, Cristina; Henriques, Ricardo; Gardner, Rui; Sepúlveda, Nuno; Simões, Pedro D; Raquel, Helena; Almeida, António Paulo; Moita, Luis F; Frischknecht, Friedrich; Mota, Maria M
Plasmodium sporozoites are transmitted by Anopheles mosquitoes and infect hepatocytes, where a single sporozoite replicates into thousands of merozoites inside a parasitophorous vacuole. The nature of the Plasmodium-host cell interface, as well as the interactions occurring between these two organisms, remains largely unknown. Here we show that highly dynamic hepatocyte actin reorganization events occur around developing Plasmodium berghei parasites inside human hepatoma cells. Actin reorganization is most prominent between 10 to 16 hours post infection and depends on the actin severing and capping protein, gelsolin. Live cell imaging studies also suggest that the hepatocyte cytoskeleton may contribute to parasite elimination...
Stankovic, Ana; Jansen, Lars E.T.
The kinetochore forms the site of attachment for mitotic spindle microtubules driving chromosome segregation. The interdependent protein interactions in this large structure have made it difficult to dissect the function of its components. In this issue, Hori et al. (2013. J. Cell Biol. http://dx.doi.org/10.1083/jcb.201210106) present a novel and powerful methodology to address the sufficiency of individual proteins for the creation of a functional de novo centromere.
Mata, João F.; Lopes, Telma; Gardner, Rui; Jansen, Lars E. T.
Gene targeting protocols for mammalian cells remain inefficient and labor intensive. Here we describe FASTarget, a rapid, fluorescent cell sorting based strategy to isolate rare gene targeting events in human somatic cells. A fluorescent protein is used as a means for direct selection of targeted clones obviating the need for selection and outgrowth of drug resistant clones. Importantly, the use of a promoter-less, ATG-less construct greatly facilitates the recovery of correctly targeted cells. Using this method we report successful gene targeting in up to 94% of recovered human somatic cell clones. We create functional EYFP-tagged knockin clones in both transformed...