Use they are in a position to separate the two daughter nuclei solely by pulling forces exerted via astral microtubules, most like via minus-end directed motor activity of cortical dynein [237]. four. Centrosome-Nucleus Attachment Like all centrosomal structures in Rigosertib Protocol vegetative cells, the Dictyostelium centrosome is structurally linked to the cytosolic side with the nucleus during interphase. Not surprisingly, one particular important protein of this linkage is definitely the nuclear envelope protein Sun1, named right after the founding members of the Sun-family, i.e., fission yeast Sad1 and Caenorhabditis elegans UNC-84, which share a typical Sun-domain. In most eukaryotes Sun1 is an inner nuclear membrane protein, forming a trimer and interacting, by way of its Sun-domain, with all the so-called KASH-domain proteins (named just after Klarsicht, ANC-1, SYNE1 homology) within the perinuclear space [239]. Since the numerous KASH domain proteins interact straight or indirectly with all 3 cytoskeletal components (actin, microtubules, intermediate filaments) the term LINC complex (linker in the nucleus and cytoskeleton) was coined for the Sun/KASH domain protein heterodimer [240]. At the nuclear side, Sun1 interacts with lamins in animal cells and also in Dictyostelium [241]. However, around the cytosolic face on the nuclear envelope the situation in Dictyostelium seems to be unique. Sun1 is present in both nuclear membanes with no sturdy bias towards the inner nuclear membrane [124,125] and there is absolutely no clear orthologue for a KASH domain protein. As a result of its similarity to mammalian nesprins, the outer nuclear membrane protein interaptin was discussed as a Dictyostelium KASH domain protein [125,242]. But interaptin is absolutely no aspect of a LINC complex, as it lacks the conserved KASH domain and obviously will not interact with Sun1 [125]. Sun1 is Xanthoangelol Monoamine Oxidase nonetheless expected for centrosome/nucleus attachment. It co-purifies with isolated centrosomes and is concentrated in the nuclear envelope within the direct vicinity from the centrosome (Figure four). Sun1 mutants are defective in centrosome/nucleus attachment. It really is possible that the centrosome/nucleus linker employs Sun1 on each sides from the membrane, and that an unknown protein in the perinuclear space mediates this interaction. Although a direct interaction with Sun1 remains to be established, the uncommon kinesin Kif9 is actually a most likely candidate for any LINC complex element in Dictyostelium. Kif9 is an internal motor kinesin, which is often grouped into the kinesin-13 family members, which usually act as microtubule depolymerases [130]. Within this group Kif9 is one of a kind in containing a 23 residue transmembrane domain close to its C-terminal end, targeting the protein to the outer nuclear envelope exactly where it accumulates within the pericentrosomal area. Knockout of Kif9 disrupts the centrosome/nucleus linkage and causes dispersal of Sun1, away in the pericentrosomal area of your nuclear envelope [130].Figure 4. Centrosome-Nucleus-Centromere cluster. (A) Immunoelectron microscopy image showing a single section of an isolated nucleus with all the attached centrosome. Nuclei have been labeled with an antibody against Dictyostelium Sun1 and nanogold conjugated anti-rabbit antibodies. The centrosome (Cn), the centromeric cluster (Cm), the nuclear envelope (NE) and also the endoplasmic reticulum (ER) are indicated (image by Prof. Otto Baumann); (B) Immunofluorescence microscopy image of a Sun1-GFP knock-in cell (green) stained with an antibody against the centrosomal core protein CP91 and anti-rabbit-AlexaFluor 568 conjug.