ABSTRACT
Mammalian neuroepithelial stem cells divide using a polarized form of cytokinesis, which is not well understood. The cytokinetic furrow cleaves the cell by ingressing from basal to apical, forming the midbody at the apical membrane. The midbody mediates abscission by recruiting many factors, including the Kinesin-6 family member Kif20b. In developing embryos, Kif20b mRNA is most highly expressed in neural stem/progenitor cells. A loss-of-function mutant in Kif20b, magoo, was found in a forward genetic screen. magoo has a small cerebral cortex, with reduced production of progenitors and neurons, but preserved layering. In contrast to other microcephalic mouse mutants, mitosis and cleavage furrows of cortical stem cells appear normal in magoo. However, apical midbodies show changes in number, shape and positioning relative to the apical membrane. Interestingly, the disruption of abscission does not appear to result in binucleate cells, but in apoptosis. Thus, Kif20b is required for proper midbody organization and abscission in polarized cortical stem cells and has a crucial role in the regulation of cerebral cortex growth.
Subject(s)
Cerebral Cortex/metabolism , Cytokinesis/physiology , Kinesins/metabolism , Neural Stem Cells/metabolism , Animals , Cell Polarity/genetics , Gene Expression , Kinesins/genetics , Mice , Mice, Inbred C57BL , Microtubules/metabolism , RNA, Messenger/biosynthesisABSTRACT
Cytoplasmic dynein is the multisubunit motor protein for retrograde movement of diverse cargoes to microtubule minus ends. Here, we investigate the function of dynein variants, defined by different intermediate chain (IC) isoforms, by expressing fluorescent ICs in neuronal cells. Green fluorescent protein (GFP)-IC incorporates into functional dynein complexes that copurify with membranous organelles. In living PC12 cell neurites, GFP-dynein puncta travel in both the anterograde and retrograde directions. In cultured hippocampal neurons, neurotrophin receptor tyrosine kinase B (TrkB) signaling endosomes are transported by cytoplasmic dynein containing the neuron-specific IC-1B isoform and not by dynein containing the ubiquitous IC-2C isoform. Similarly, organelles containing TrkB isolated from brain by immunoaffinity purification also contain dynein with IC-1 but not IC-2 isoforms. These data demonstrate that the IC isoforms define dynein populations that are selectively recruited to transport distinct cargoes.
Subject(s)
Cytoplasm/metabolism , Dyneins/metabolism , Endosomes/enzymology , Neurons/metabolism , Receptor, trkB/metabolism , Signal Transduction , Animals , Cells, Cultured , Cytoplasm/drug effects , Endosomes/drug effects , Green Fluorescent Proteins/metabolism , Kinetics , Microtubules/drug effects , Microtubules/metabolism , Nerve Growth Factors/pharmacology , Neurites/drug effects , Neurites/metabolism , Neurons/cytology , Neurons/drug effects , Organ Specificity/drug effects , PC12 Cells , Protein Isoforms/metabolism , Protein Transport/drug effects , RNA, Small Interfering/metabolism , Rats , Receptor, trkA/metabolism , Recombinant Fusion Proteins/metabolism , Signal Transduction/drug effectsABSTRACT
The cytoplasmic dynein 1 cargo binding domain is formed by five subunits including the intermediate chain and the DYNLT, DYNLL, and DYNLRB light chain families. Six isoforms of the intermediate chain and two isoforms of each of the light chain families have been identified in mammals. There is evidence that different subunit isoforms are involved in regulating dynein function, in particular linking dynein to different cargoes. However, it is unclear how the subunit isoforms are assembled or if there is any specificity to their interactions. Co-immunoprecipitation using DYNLT-specific antibodies reveals that dynein complexes with DYNLT light chains also contain the DYNLL and DYNLRB light chains. The DYNLT light chains, but not DYNLL light chains, associate exclusively with the dynein complex. Yeast two-hybrid and co-immunoprecipitation assays demonstrate that both members of the DYNLT family are capable of forming homodimers and heterodimers. In addition, both homodimers of the DYNLT family bind all six intermediate chain isoforms. However, DYNLT heterodimers do not bind to the intermediate chain. Thus, whereas all combinations of DYNLT light chain dimers can be made, not all of the possible combinations of the isoforms are utilized during the assembly of the dynein complex.
