Use they are able to separate the two daughter nuclei solely by pulling forces exerted by means of astral microtubules, most like by way of minus-end directed motor activity of cortical dynein [237]. four. Centrosome-Nucleus Attachment Like all centrosomal structures in vegetative cells, the Dictyostelium centrosome is structurally linked for the cytosolic side of your nucleus in the course of interphase. Not surprisingly, one particular important protein of this linkage is definitely the Olutasidenib Purity & Documentation nuclear envelope protein Sun1, named following the founding members in 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, via its Sun-domain, using the so-called KASH-domain proteins (named right after Klarsicht, ANC-1, SYNE1 homology) inside the perinuclear space [239]. Since the several KASH domain proteins interact straight or indirectly with all 3 cytoskeletal components (actin, microtubules, intermediate filaments) the term LINC complex (linker from the nucleus and cytoskeleton) was coined for the Sun/KASH domain protein heterodimer [240]. In the nuclear side, Sun1 interacts with lamins in animal cells as well as in Dictyostelium [241]. But, on the cytosolic face of the nuclear envelope the circumstance in Dictyostelium appears to become exclusive. Sun1 is present in both nuclear membanes with no strong bias towards the inner nuclear membrane [124,125] and there is absolutely no clear orthologue to get 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 undoubtedly no element of a LINC complicated, as it lacks the conserved KASH domain and obviously does not interact with Sun1 [125]. Sun1 is having said that needed for centrosome/nucleus attachment. It co-purifies with isolated centrosomes and is concentrated at the nuclear envelope within the direct vicinity in the centrosome (Figure four). Sun1 mutants are defective in centrosome/nucleus attachment. It really is probable that the centrosome/nucleus linker employs Sun1 on both sides of your membrane, and that an unknown protein with the perinuclear space mediates this interaction. Despite the fact that a direct interaction with Sun1 remains to be established, the uncommon Chelerythrine In Vivo kinesin Kif9 is actually a probably candidate for any LINC complicated element in Dictyostelium. Kif9 is an internal motor kinesin, which can be grouped in to the kinesin-13 household, which generally act as microtubule depolymerases [130]. Inside this group Kif9 is unique in containing a 23 residue transmembrane domain close to its C-terminal end, targeting the protein to the outer nuclear envelope where it accumulates inside the pericentrosomal area. Knockout of Kif9 disrupts the centrosome/nucleus linkage and causes dispersal of Sun1, away in the pericentrosomal region with the nuclear envelope [130].Figure four. Centrosome-Nucleus-Centromere cluster. (A) Immunoelectron microscopy image showing a single section of an isolated nucleus with the attached centrosome. Nuclei were 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 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.