The distinct roles of the nucleus and nucleus-cytoskeleton connections in three-dimensional cell migration

Cells often migrate in vivo in an extracellular matrix that is intrinsically three-dimensional (3D) and the role of actin filament architecture in 3D cell migration is less well understood. Here we show that, while recently identified linkers of nucleoskeleton to cytoskeleton (LINC) complexes play a minimal role in conventional 2D migration, they play a critical role in regulating the organization of a subset of actin filament bundles – the perinuclear actin cap - connected to the nucleus through Nesprin2giant and Nesprin3 in cells in 3D collagen I matrix. Actin cap fibers prolong the nucleus and mediate the formation of pseudopodial protrusions, which drive matrix traction and 3D cell migration. Disruption of LINC complexes disorganizes the actin cap, which impairs 3D cell migration. A simple mechanical model explains why LINC complexes and the perinuclear actin cap are essential in 3D migration by providing mechanical support to the formation of pseudopodial protrusions

KIFC1-Like Motor Protein Associates with the Cephalopod Manchette and Participates in Sperm Nuclear Morphogenesis in Octopus tankahkeei

Nuclear morphogenesis is one of the most fundamental cellular transformations taking place during spermatogenesis. In rodents, a microtubule-based perinuclear structure, the manchette, and a C-terminal kinesin motor KIFC1 are believed to play crucial roles in this process. Spermatogenesis in Octopus tankahkeei is a good model system to explore whether evolution has created a cephalopod prototype of mammalian manchette-based and KIFC1-dependent sperm nuclear shaping machinery.We detected the presence of a KIFC1-like protein in the testis, muscle, and liver of O. tankahkeei by Western Blot. Then we tracked its dynamic localization in spermatic cells at various stages using Immunofluorescence and Immunogold Electron Microscopy. The KIFC1-like protein was not expressed at early stages of spermatogenesis when no significant morphological changes occur, began to be present in early spermatid, localized around and in the nucleus of intermediate and late spermatids where the nucleus was dramatically elongated and compressed, and concentrated at one end of final spermatid. Furthermore, distribution of the motor protein during nuclear elongation and condensation overlapped with that of the cephalopod counterpart of manchette at a significant level.The results support the assumption that the protein is actively involved in sperm nuclear morphogenesis in O. tankahkeei possibly through bridging the manchette-like perinuclear microtubules to the nucleus and assisting in the nucleocytoplasmic trafficking of specific cargoes. This study represents the first description of the role of a motor protein in sperm nuclear shaping in cephalopod

The SUN Protein Mps3 Is Required for Spindle Pole Body Insertion into the Nuclear Membrane and Nuclear Envelope Homeostasis

The budding yeast spindle pole body (SPB) is anchored in the nuclear envelope so that it can simultaneously nucleate both nuclear and cytoplasmic microtubules. During SPB duplication, the newly formed SPB is inserted into the nuclear membrane. The mechanism of SPB insertion is poorly understood but likely involves the action of integral membrane proteins to mediate changes in the nuclear envelope itself, such as fusion of the inner and outer nuclear membranes. Analysis of the functional domains of the budding yeast SUN protein and SPB component Mps3 revealed that most regions are not essential for growth or SPB duplication under wild-type conditions. However, a novel dominant allele in the P-loop region, MPS3-G186K, displays defects in multiple steps in SPB duplication, including SPB insertion, indicating a previously unknown role for Mps3 in this step of SPB assembly. Characterization of the MPS3-G186K mutant by electron microscopy revealed severe over-proliferation of the inner nuclear membrane, which could be rescued by altering the characteristics of the nuclear envelope using both chemical and genetic methods. Lipid profiling revealed that cells lacking MPS3 contain abnormal amounts of certain types of polar and neutral lipids, and deletion or mutation of MPS3 can suppress growth defects associated with inhibition of sterol biosynthesis, suggesting that Mps3 directly affects lipid homeostasis. Therefore, we propose that Mps3 facilitates insertion of SPBs in the nuclear membrane by modulating nuclear envelope composition

LINC-anchored actin cap connects the extracellular milieu to the nucleus for ultrafast mechanotransduction

PMC3548190Cells continuously sense and respond to external mechanical forces through their cytoskeleton. Here we show that only a small subset of actin fibers, those forming the perinuclear actin cap that wraps around the nucleus, form in response to low physiological mechanical stresses in adherent fibroblasts. While conventional basal stress fibers form only past a threshold shear stress of 0.5 dyn/cm(2), actin-cap fibers are formed at shear stresses 50 times lower and orders-of-magnitude faster than biochemical stimulation. This fast differential response is uniquely mediated by focal adhesion protein zyxin at low shear stress and actomyosin fibers of the actin cap. We identify additional roles for lamin A/C of the nuclear lamina and linkers of nucleus to cytoskeleton (LINC) molecules nesprin2giant and nesprin3, which anchor actin cap fibers to the nucleus. These results suggest an interconnected physical pathway for mechanotransduction, from the extracellular milieu to the nucleus.JH Libraries Open Access Fun

The plant LINC complex at the nuclear envelope

Significant advances in understanding the plant nuclear envelope have been made over the past few years; indeed, knowledge of the protein network at the nuclear envelope is rapidly growing. One such network, the linker of nucleoskeleton and cytoskeleton (LINC) complex, is known in animals to connect chromatin to the cytoskeleton through the nuclear envelope. The LINC complex is made of Sad1/Unc84 (SUN) and Klarsicht/Anc1/Syne1 homology (KASH) proteins which have been recently characterized in plants. SUN proteins are located within the inner nuclear membrane, while the KASH proteins are included into the outer nuclear membrane. SUN and KASH domains interact and bridge the two nuclear membranes. In Arabidopsis, KASH proteins also interact with the tryptophan-proline-proline (WPP) domain-interacting tail-anchored protein 1 (WIT1), associated with the nuclear pore complex and with myosin XI-i which directly interacts with the actin cytoskeleton. Although evidence for a plant LINC complex connecting the nucleus to the cytoskeleton is growing, its interaction with chromatin is still unknown, but knowledge gained from animal models strongly suggests its existence in plants. Possible functions of the plant LINC complex in cell division, nuclear shape, and chromatin organization are discussed

Analyses Of Dynein Heavy Chain Mutations Reveal Complex Interactions Between Dynein Motor Domains And Cellular Dynein Functions

Cytoplasmic dynein transports cargoes for a variety of crucial cellular functions. However, since dynein is essential in most eukaryotic organisms, the in-depth study of the cellular function of dynein via genetic analysis of dynein mutations has not been practical. Here, we identify and characterize 34 different dynein heavy chain mutations using a genetic screen of the ascomycete fungus Neurospora crassa, in which dynein is nonessential. Interestingly, our studies show that these mutations segregate into five different classes based on the in vivo localization of the mutated dynein motors. Furthermore, we have determined that the different classes of dynein mutations alter vesicle trafficking, microtubule organization, and nuclear distribution in distinct ways and require dynactin to different extents. In addition, biochemical analyses of dynein from one mutant strain show a strong correlation between its in vitro biochemical properties and the aberrant intracellular function of that altered dynein. When the mutations were mapped to the published dynein crystal structure, we found that the three-dimensional structural locations of the heavy chain mutations were linked to particular classes of altered dynein functions observed in cells. Together, our data indicate that the five classes of dynein mutations represent the entrapment of dynein at five separate points in the dynein mechanochemical and transport cycles. We have developed N. crassa as a model system where we can dissect the complexities of dynein structure, function, and interaction with other proteins with genetic, biochemical, and cell biological studies. © 2012 by the Genetics Society of America

Analyses of Dynein Heavy Chain Mutations Reveal Complex Interactions Between Dynein Motor Domains and Cellular Dynein Functions

Cytoplasmic dynein transports cargoes for a variety of crucial cellular functions. However, since dynein is essential in most eukaryotic organisms, the in-depth study of the cellular function of dynein via genetic analysis of dynein mutations has not been practical. Here, we identify and characterize 34 different dynein heavy chain mutations using a genetic screen of the ascomycete fungus Neurospora crassa, in which dynein is nonessential. Interestingly, our studies show that these mutations segregate into five different classes based on the in vivo localization of the mutated dynein motors. Furthermore, we have determined that the different classes of dynein mutations alter vesicle trafficking, microtubule organization, and nuclear distribution in distinct ways and require dynactin to different extents. In addition, biochemical analyses of dynein from one mutant strain show a strong correlation between its in vitro biochemical properties and the aberrant intracellular function of that altered dynein. When the mutations were mapped to the published dynein crystal structure, we found that the three-dimensional structural locations of the heavy chain mutations were linked to particular classes of altered dynein functions observed in cells. Together, our data indicate that the five classes of dynein mutations represent the entrapment of dynein at five separate points in the dynein mechanochemical and transport cycles. We have developed N. crassa as a model system where we can dissect the complexities of dynein structure, function, and interaction with other proteins with genetic, biochemical, and cell biological studies

Multiple-motor based transport and its regulation by Tau

Motor-based intracellular transport and its regulation are crucial to the functioning of a cell. Disruption of transport is linked to Alzheimer's and other neurodegenerative diseases. However, many fundamental aspects of transport are poorly understood. An important issue is how cells achieve and regulate efficient long-distance transport. Mounting evidence suggests that many in vivo cargoes are transported along microtubules by more than one motor, but we do not know how multiple motors work together or can be regulated. Here we first show that multiple kinesin motors, working in conjunction, can achieve very long distance transport and apply significantly larger forces without the need of additional factors. We then demonstrate in vitro that the important microtubule-associated protein, tau, regulates the number of engaged kinesin motors per cargo via its local concentration on microtubules. This function of tau provides a previously unappreciated mechanism to regulate transport. By reducing motor reattachment rates, tau affects cargo travel distance, motive force, and cargo dispersal. We also show that different isoforms of tau, at concentrations similar to those in cells, have dramatically different potency. These results provide a well defined mechanism for how altered tau isoform levels could impair transport and thereby lead to neurodegeneration without the need of any other pathway

Lamin B1 and lamin B2 are long-lived proteins with distinct functions in retinal development

Lamin B1 and lamin B2 are essential building blocks of the nuclear lamina, a filamentous meshwork lining the nucleoplasmic side of the inner nuclear membrane. Deficiencies in lamin B1 and lamin B2 impair neurodevelopment, but distinct functions for the two proteins in the development and homeostasis of the CNS have been elusive. Here we show that embryonic depletion of lamin B1 in retinal progenitors and postmitotic neurons affects nuclear integrity, leads to the collapse of the laminB2 meshwork, impairs neuronal survival, and markedly reduces the cellularity of adult retinas. In stark contrast, a deficiency of lamin B2 in the embryonic retina has no obvious effect on lamin B1 localization or nuclear integrity in embryonic retinas, suggesting that lamin B1, but not lamin B2, is strictly required for nucleokinesis during embryonic neurogenesis. However, the absence of lamin B2 prevents proper lamination of adult retinal neurons, impairs synaptogenesis, and reduces cone photoreceptor survival. We also show that lamin B1 and lamin B2 are extremely long-lived proteins in rod and cone photoreceptors. OF interest, a complete absence of both proteins during postnatal life has little or no effect on the survival and function of cone photoreceptors