Ectosome biogenesis and release processes observed by using live-cell dynamic imaging in mammalian glial cells.

BACKGROUND: Ectosomes are recognized as shedding from the plasma membranes into the extracellular environment. Recent research has demonstrated that ectosomes are surrounded by phospholipid membranes containing lipid rafts and caveolae. Some ectosomes contain cytokines in the lumen and have high levels of phosphatidylserine exposed to the outer membrane. Intracellular vesicles share both characters with ectosomes. Why the plasma membrane-derived ectosomes have the same characteristics as intracellular vesicles remain largely unknown. METHODS: Using live-cell dynamic imaging, we recorded the process of ectosome biogenesis and release in primary cultured neural cells. RESULTS: Our results show two different ectosome release methods: slow-releasing and fast-releasing. In the slow-releasing, multiple ectosomes emerge almost simultaneously on the cell surface and are released by outward budding from the plasma membrane. In the fast releasing, ectosomes squeeze out of the membrane domain and pinch off from a cell's surface. Using ER-tracker for live-cell imaging, we directly observed the process that intracellular vesicles jump out of the plasma membrane for release. This type of ectosomes has a reverse array of membrane proteins and phospholipids compared to the plasma membrane. So ectosomes should be divided into two groups: plasma membrane-derived and intracellular membrane-derived ectosomes. CONCLUSIONS: Both slow releasing and fast releasing EVs imply mechanisms of human diseases and for diagnostics and drug delivery.

Leaching Characteristics of Heavy Metals and Plant Nutrients in the Sewage Sludge Immobilized by Composite Phosphorus-Bearing Materials.

In order to evaluate the environmental risk caused by land application of sewage sludge, leaching characteristics of heavy metals and plant nutrients in the sewage sludge immobilized by composite phosphorus-bearing materials were investigated. Their cumulative release characteristics were confirmed. Furthermore, the first-order kinetics equation, modified Elovich equation, double-constant equation, and parabolic equation were used to explore dynamic models of release. Results showed that sewage sludge addition significantly increased electricity conductivity (EC) in leachates, and the concentrations of heavy metals (Cu, Cr, Zn) and plant nutrients (N, P, K) were also obviously increased. The highest concentrations of Cu, Cr, and Zn in the leachates were all below the limit values of the fourth level in the Chinese national standard for groundwater quality (GB/T14848-2017). The immobilization of composite phosphorus-bearing materials reduced the release of Cu and Cr, while increased that of Zn. The fitting results of modified Elovich model and double-constant model were in good agreement with the leaching process of heavy metals and plant nutrients, indicating their release process in soil under simulated leaching conditions was not a simple first-order reaction, but a complex heterogeneous diffusion process controlled by multifactor.

Anti-influenza virus effects of crude phenylethanoid glycosides isolated from ligustrum purpurascens via inducing endogenous interferon-γ.

ETHNOPHARMACOLOGICAL RELEVANCE: Ligustrum purpurascens Y.C. Yang (Oleaceae) is traditionally recorded as "Ku Ding Cha", a kind of functional tea in southern China for about two thousand years, which has been reported with sore throat alleviating and pathogenic heat expelling effects. However, there are no scientific studies demonstrating its antiviral activity. THE AIM OF THE STUDY: This study is aimed at investigating the anti-influenza virus effects of phenylethanoid glycosides isolated from L. purpurascens (LPG) as well as its corresponding mechanisms. MATERIALS AND METHODS: In vitro, hemagglutination assay was employed to detect the influenza virus titer; In vivo, C57BL/6J mice were given oral administration of LPG (100mg/kg, 300mg/kg, 900mg/kg) or ribavirin (100mg/kg) once daily for 5 successive days. Meanwhile, on the second day, mice were infected intranasally (i.n.) with A/FM/1/47 H1N1 virus. Mice survival rate and other clinical index were monitored for 15 days. Infected mice were sacrificed to measure the lung lesion and stained with hematoxylin-eosin. Flow cytometry analyses spleen lymphocytes and interferon-γ (IFN-γ) level. The IFN-γ knockout mice (IFN-γ(-/-) mice, C57BL/6J) which had been verified lacking IFN-γ through Western Blot, were applied in the death-protection test to identify the role of IFN-γ played in LPG antiviral effect. RESULTS: In vitro, LPG at 0.5mg/ml inhibited Influenza A Virus H1N1 type (H1N1) infection of MDCK cells. In vivo, LPG at 300 and 900mg/kg significantly decreased the mouse lung index (p<0.05), alleviated influenza-induced lethality and clinical symptoms, and therefore enhanced mouse survival (p<0.05). More detailed experiments demonstrated that antiviral cytokine IFN-γ was involved in the antiviral effect of LPG. Flow cytometric analysis revealed that LPG (900mg/kg) significantly induced secretion of IFN-γ by splenic CD4(+) and CD8(+) cells (p<0.05). Moreover, LPG (900mg/kg) protected wild-type C57BL/6J mice from H1N1 injury, whereas LPG-mediated survival protection disappeared in IFN-γ(-/-) mice. CONCLUSION: These results suggest that up-regulating endogenous IFN-γ by LPG may represent a novel therapeutic approach for H1N1 infection.

A novel tissue-engineered bone in repairing femoral head defect and necrosis.

OBJECTIVE: To evaluate the therapeutic effects of AACB/BMP/bFGF, a novel tissue-engineered bone, in repairing femoral head defect and necrosis in dog models. METHODS: Dog models of avascular necrosis of femoral head (ANFH) were established by liquid nitrogen freezing method. Group A was untreated; Groups B, C, and D were implanted with AACB, AACB/BMP, and AACB/BMP/bFGF complex, respectively; Group E was grafted with autologous cancellous bone. Samples were collected at 3 w, 6 w, and 12 w after operation. A series of examinations were carried out to investigate the effects of the materials in repairing femoral head defect, including anatomical observation, X-ray examination, histological analysis, and vascular immunohistochemical staining. RESULTS: Our results indicated that, compared with AACB alone and AACB/BMP, AACB/BMP/bFGF complex could exert the most efficient therapeutic effects in dog ANFH models. X-ray examination further confirmed that AACB/BMP/bFGF complex could effectively repair the injuries in dog ANFH models, almost to a comparable level with cancellous bone autografts. Moreover, histological analysis indicated that AACB/BMP/bFGF complex greatly enhanced the new bone formation, which would contribute to the healing of ANFH. Furthermore, vascular immunohistochemical staining revealed that AACB/BMP/bFGF complex could significantly stimulate the revascularization in defect areas, reflecting the post-injury healing process in these models. CONCLUSION: AACB/BMP/bFGF complex has great potential in repairing femoral head defect by enhancing osteogenesis and revascularization. The novel tissue-engineered bone would be widely used in clinical applications for ANFH treatment, especially as an alternative for autografts.

Propidium iodide (PI) stains Nissl bodies and may serve as a quick marker for total neuronal cell count.

Propidium iodide (PI) reacts with both DNA and RNA and is a commonly used fluorescent reagent for nucleic acid staining. The aim of the study was to compare the cellular staining patterns of PI with that of Nissl staining in rat nervous tissues and to report a modified staining method that selectively labels Nissl bodies in neurons. Cryosections and paraffin sections of different tissues of normal Sprague-Dawley rats, including trigeminal ganglia, dorsal root ganglia, spinal cord, liver, and small intestine, were stained by either PI or the hematoxylin and eosin method. Some sections were treated with RNase or DNase before the above staining, and some were double stained with PI and a Nissl stain. The sections were observed by light, fluorescence or confocal microscopy. Results showed strong PI signals detected as patterns of granules in the neuronal cytoplasm of all nervous tissues, whereas the staining of neuronal nuclei was weaker. In contrast, nuclei of neuroglial cells were strongly stained by PI, while the cytoplasm was not obviously stained. Pretreatment of the neural tissue with RNase abolished the PI signals. Furthermore, the PI positive granules in neuronal cytoplasm co-localized with Nissl bodies stained by the fluorescent Nissl stain. When the tissue was pretreated with DNase, PI only stained the cytoplasmic granules of neurons, but not that of glial cells. Our results show that PI stains Nissl bodies and may serve as an economical and convenient neuron marker for neuronal cell counting when specific neural markers such as antibodies are not readily available.

Dual-index evaluation of character changes in Panax ginseng C. A. Mey stored in different conditions.

Panax ginseng C. A. Mey has been used as a traditional medicine and functional food in Asia for thousands of years for its improvement of human immunity and metabolism and its antitumor and antifatigue activities. This study reports the impact of storage conditions and storage period on the quality of P. ginseng. The contents of four major ginsenosides in P. ginseng and phosphorylation activities of Akt of ginseng extracts were affected by both storage conditions and storage period. In contrast, the ATP generation capacity of ginseng extracts was affected by storage conditions, but not by storage period. The results showed that the quality of P. ginseng could be well maintained at a relative humidity between 70% and 90%, and dry conditions might decrease the quality of P. ginseng. Through dual-index evaluation, the present study extended our knowledge on the changes of ginsenosides and bioactivities in P. ginseng with respect to different storage conditions and storage periods.

Involvement of ERK1/2, cPLA2 and NF-κB in microglia suppression by cannabinoid receptor agonists and antagonists.

Cannabinoids have been consistently shown to suppress microglia activation and the release of cytotoxic factors including nitric oxide, superoxide and proinflammatory cytokines. However, the underlying molecular mechanisms and whether the action of cannabinoids is coupled to the activation of cannabinoid type 1 (CB1) and type 2 (CB2) receptors are still poorly defined. In this study we observed that the CB1 and CB2 receptor non-selective or selective agonists dramatically attenuate iNOS induction and ROS generation in LPS-activated microglia. These effects are due to their reduction of phosphorylation of extracellular signal regulated kinase 1/2 (ERK1/2), cytosolic phospholipase A (cPLA) and activation of NF-κB. Surprisingly, instead of reversing the effect of the respective CB1 and CB2 receptor agonists, the antagonists also suppress iNOS induction and ROS generation in activated microglia by similar mechanisms. Taken together, these results indicate that both cannabinoid receptor agonists and antagonists might suppress microglia activation by CB1 and CB2 receptor independent mechanisms, and provide a new insight into the mechanisms of microglia inhibition by cannabinoids.

Arachidonyl trifluoromethyl ketone ameliorates experimental autoimmune encephalomyelitis via blocking peroxynitrite formation in mouse spinal cord white matter.

Inhibition of phospholipase A(2) (PLA(2)) has recently been found to attenuate the pathogenesis of experimental autoimmune encephalomyelitis (EAE), a commonly used animal model of multiple sclerosis (MS). However, the protective mechanisms that underlie PLA(2) inhibition are still not well understood. In this study, we found that cytosolic PLA(2) (cPLA(2)) was highly expressed in infiltrating lymphocytes and macrophages/microglia in mouse spinal cord white matter. Although cPLA(2) is also expressed in spinal cord neurons and oligodendrocytes, there were no differences observed in these cell types between EAE and control animals. Arachidonyl trifluoromethyl ketone (AACOCF3), a cPLA(2) inhibitor, significantly reduced the clinical symptoms and inhibited the body weight loss typically found in EAE mice. AACOCF3 also attenuated the loss of mature, myelin producing, oligodendrocytes, and axonal damage in the spinal cord white matter. Nitrotyrosine immunoreactivity, an indicator of peroxynitrite formation, was dramatically increased in EAE mice and attenuated by treatment with AACOCF3. These protective effects were not evident when AA861, an inhibitor of lipoxygenase, was used. In primary cultures of microglia, lipopolysaccharide (LPS) induced an upregulation of cPLA(2), inducible nitric oxide synthase (iNOS) and components of the NADPH oxidase complex, p47phox and p67phox. AACOCF3 significantly attenuated iNOS induction, nitric oxide production and the generation of reactive oxygen species in reactive microglia. Similar to the decomposition catalyst of peroxynitrite, AACOCF3 also blocked oligodendrocyte toxicity induced by reactive microglia. These results suggest that AACOCF3 may prevent oligodendrocyte loss in EAE by attenuating peroxynitrite formation in the spinal cord white matter.

Protein kinase C mediates peroxynitrite toxicity to oligodendrocytes.

Peroxynitrite has been suggested to be the potent oxidant causing toxicity to neurons and oligodendrocytes (OLs). Our previous studies have illustrated that intracellular zinc liberation contributes to peroxynitrite toxicity to mature OLs. In this study, we further investigated the signaling pathways involved in this event and identified protein kinase C (PKC) as an important early signaling molecule. We found that a non-selective PKC inhibitor bisindolylmaleimide-1 blocked OL toxicity induced by a peroxynitrite generator SIN-1 and exogenous zinc. The protective effects were due to its inhibition on ERK1/2 phosphorylation and ROS generation. The same phenomenon was also observed in OLs following prolonged treatment with phorbol 12 myristate 13 acetate (PMA), which downregulates the conventional and the novel PKC isoforms (cPKCs and nPKCs). To determine the role of specific PKC isoforms, we found that a specific nPKC inhibitor rottlerin significantly reduced SIN-1- or zinc-induced toxicity, whereas Go6976, a cPKC inhibitor, reduced OL toxicity triggered by zinc, but not by SIN-1 at high concentrations. Rottlerin was more potent than Go6976 to attenuate ERK1/2 phosphorylation and ROS generation induced by SIN-1 or zinc. Surprisingly, zinc only induced phosphorylation of PKCθ, but not PKCδ. Knockdown of PKCθ using lentiviral shRNA attenuated SIN-1- or zinc-induced toxicity. These results suggest that PKCθ might be the major PKC isoform involved in peroxynitrite and zinc toxicity to mature OLs, and provide a rationale for development of specific inhibitors of PKCθ in the treatment of multiple sclerosis and other neurodegenerative diseases, in which peroxynitrite formation plays a pathogenic role.

Dynein light intermediate chain in Aspergillus nidulans is essential for the interaction between heavy and intermediate chains.

Cytoplasmic dynein is a complex containing heavy chains (HCs), intermediate chains (ICs), light intermediate chains (LICs), and light chains (LCs). The HCs are responsible for motor activity. The ICs at the tail region of the motor interact with dynactin, which is essential for dynein function. However, functions of other subunits and how they contribute to the assembly of the core complex are not clearly defined. Here, we analyzed in the filamentous fungus Aspergillus nidulans functions of the only LIC and two LCs, RobA (Roadblock/LC7) and TctexA (Tctex1) in dynein-mediated nuclear distribution (nud). Whereas the deletion mutant of tctexA did not exhibit an apparent nud mutant phenotype, the deletion mutant of robA exhibited a nud phenotype at an elevated temperature, which is similar to the previously characterized nudG (LC8) deletion mutant. Remarkably, in contrast to the single mutants, the robA and nudG double deletion mutant exhibits a severe nud phenotype at various temperatures. Thus, functions of these two LC classes overlap to some extent, but the presence of both becomes important under specific conditions. The single LIC, however, is essential for dynein function in nuclear distribution. This is evidenced by the identification of the nudN gene as the LIC coding gene, and by the nud phenotype exhibited by the LIC down-regulating mutant, alcA-LIC. Without a functional LIC, the HC-IC association is significantly weakened, and the HCs could no longer accumulate at the microtubule plus end. Thus, the LIC is essential for the assembly of the core complex of dynein in Aspergillus.

[Total hip arthroplasty for the treatment of developmental dysplasia of the hip in adults].

OBJECTIVE: To summarize techniques of the total hip arthroplasty (THA) in the treatment of developmental dysplasia of the hip (DDH) with severe osteoarthritis in adults. METHODS: From March 2000 to January 2006, 24 patients (27 hips) with DDH were treated by THA with an cementless cup. There were 7 males and 17 females, with the average age of 49.6 years (ranging from 26 years to 63 years). Unilateral DDH occurred in 21 patients and bilateral DDH occurred in 3 patients. Based on the Crowe classification, there were 16 hips in 15 patients of type I, 4 hips in 4 patients of type II, 4 hips in 3 patients of type III, 3 hips in 2 patients of type IV. Except for 3 patients with bilateral DDH, the other patients' ill lower limbs were 2-7 cm shorter than the healthy lower ones. RESULTS: All the patients were followed up from 9 months to 6.5 years and no one had infection, dislocation, femur fracture and so on after the operation. In 18 patients, the pain was completely relieved and the function of the hip joints was good. After the gluteus medius exercise, the claudication of 3 patients after the operation disappeared. In 3 patients, the ill lower limbs were more than 1 cm shorter than the healthy lower ones and the other patients' ill lower limbs were less than 1 cm shorter than the healthy lower ones. Two patients' lower limbs were been lengthened 4-5 cm. All the patients' sciatic nerves were not injured. The Harris scores were 46.5 +/- 7.2 preoperatively and 84.0 +/- 5.7 postoperatively (P < 0.05). CONCLUSION: THA with deepening the medial wall of the acetabulum at the true acetabulum and choosing small cementless cup in adult could obtain favorable results.

Arp11 affects dynein-dynactin interaction and is essential for dynein function in Aspergillus nidulans.

The dynactin complex contains proteins including p150 that interacts with cytoplasmic dynein and an actin-related protein Arp1 that forms a minifilament. Proteins including Arp11 and p62 locate at the pointed end of the Arp1 filament, but their biochemical functions are unclear (Schroer TA. Dynactin. Annu Rev Cell Dev Biol 2004;20:759-779). In Aspergillus nidulans, loss of Arp11 or p62 causes the same nuclear distribution (nud) defect displayed by dynein mutants, indicating that these pointed-end proteins are essential for dynein function. We constructed a strain with S-tagged p150 of dynactin that allows us to pull down components of the dynactin and dynein complexes. Surprisingly, while the ratio of pulled-down Arp1 to S-p150 in Arp11-depleted cells is clearly lower than that in wild-type cells, the ratio of pulled-down dynein to S-p150 is significantly higher. We further show that the enhanced dynein-dynactin interaction in Arp11-depleted cells is also present in the soluble fraction and therefore is not dependent upon the affinity of these proteins to the membrane. We suggest that loss of the pointed-end proteins alters the Arp1 filament in a way that affects the conformation of p150 required for its proper interaction with the dynein motor.

Gene expression profile related to inflammation in rat model of traumatic deep vein thrombosis.

OBJECTIVE: To study the relationship between inflammation and traumatic deep vein thrombosis (TDVT). METHODS: A rat model of deep venous thrombosis was established by directly clamping femoral vein. Based on the different biological situations of femoral vein thrombosis and observation phases, 150 SD rats were divided into 7 groups. Inflammatory cells in vein wall of each group were counted. The fold change and cluster analysis were applied to study the change of gene expression during the development of venous thrombosis. Especially, the genes related to inflammation, fibrinolysis, coagulation of endothelium were analyzed in detail. RESULTS: The inflammation cells in femoral vein wall were mostly neutrophilic granulocytes in Groups B, C and D, while they were lymphocytes in Groups E, F and G. Compared with Groups A, B, E and G, the inflammation cell counts in Groups C, D and F were much higher (P less than 0.05). The results of fold-change analysis showed that 2 504 genes (Log 2 ratio > or = 1 or < or = 1) presented different expressions in the process of TDVT. Most of these genes'functions were not clarified so far and the genes with known functions were involved in inflammation, DNA-dependent transcription regulation, blood coagulation, fibrinolysis, etc. Among them, 23 genes related to inflammation had different expressions during TDVT. The cluster analysis showed that the expression changes of several genes, such as IL-1 alpha, IL-1 beta, IL-6, Cinc2, corresponded with the development of femoral vein thrombosis. CONCLUSION: There is a close relationship between the genes related to inflammation and deep vein thrombosis induced by direct vascular trauma.

Cytoplasmic dynein's mitotic spindle pole localization requires a functional anaphase-promoting complex, gamma-tubulin, and NUDF/LIS1 in Aspergillus nidulans.

In Aspergillus nidulans, cytoplasmic dynein and NUDF/LIS1 are found at the spindle poles during mitosis, but they seem to be targeted to this location via different mechanisms. The spindle pole localization of cytoplasmic dynein requires the function of the anaphase-promoting complex (APC), whereas that of NUDF does not. Moreover, although NUDF's localization to the spindle poles does not require a fully functional dynein motor, the function of NUDF is important for cytoplasmic dynein's targeting to the spindle poles. Interestingly, a gamma-tubulin mutation, mipAR63, nearly eliminates the localization of cytoplasmic dynein to the spindle poles, but it has no apparent effect on NUDF's spindle pole localization. Live cell analysis of the mipAR63 mutant revealed a defect in chromosome separation accompanied by unscheduled spindle elongation before the completion of anaphase A, suggesting that gamma-tubulin may recruit regulatory proteins to the spindle poles for mitotic progression. In A. nidulans, dynein is not apparently required for mitotic progression. In the presence of a low amount of benomyl, a microtubule-depolymerizing agent, however, a dynein mutant diploid strain exhibits a more pronounced chromosome loss phenotype than the control, indicating that cytoplasmic dynein plays a role in chromosome segregation.

[Study on the effect of composite of basic fibroblast growth factor and partially deproteinized bone on the repair of femoral head defects].

OBJECTIVE: To evaluate the effect of composite (bFGF/PDPB) of basic fibroblast growth factor (bFGF) and partially deproteinized bone (PDPB) on the repair of femoral head defect. METHODS: Forty-eight femoral heads with defect derived from 24 New Zealand rabbits were divided into 3 groups at random, which were implanted with bFGF/PDPB (group A), PDPB (group B) and nothing (group C) respectively. The rabbits were sacrificed at 2, 4, and 8 weeks after operation, and then the femoral heads were obtained. The specimens injected with Chinese ink were created. Then X-ray examination, histopathological and morphological examination of blood vessel, and image analysis were made. RESULTS: The bone defects healed completely 8 weeks after operation in group A. The implants in the repaired tissue were not substituted completely in group B. The bone defects did not heal completely in group C. Two weeks after operation, affluent newly formed vessels were seen in repaired areas in group A. No significant difference between group A and group B was observed 8 weeks after operation. In group C, newly formed vessels were scarce 2, 4, and 8 weeks after operation. There were 3 sides rated excellent, 2 good and 1 fair in group A; 1 excellent, 2 good, 2 fair and 1 poor in group B; and 1 fair and 5 poor in group C according to the X-ray evaluation 8 weeks after operation. Eight weeks after operation, the volume fraction of bone trabecula in repaired tissue was higher in group A than that in group B (P < 0.05), and the fraction in group C was the lowest among the 3 groups (P < 0.05). CONCLUSION: The composite of bFGF and PDPB can effectively promote the repair of femoral head defect of rabbit.

[Experimental study on repairing segmental bone defects with three bio-bone derived materials].

OBJECTIVE: To evaluate the osteogenesis of three bio-bone derived materials in repairing segmental bone defects. METHODS: Sixty Japanese rabbits were made 10 mm radius segmental defects and divided into 5 groups (groups A, B, C, D and E, n=12). Composite fully deproteinised bone (CFDB, group A), partially deproteinised bone (PDPB, group B), partially decalcified bone (PDCB, group C), autogenous iliac bone graft (group D) and no implant (group E) were implanted into the radius segmental bone defects of rabbits. The specimens were examined after 4, 8, 12 and 24 weeks; the osteogenesis was evaluated through X-ray radiograph and undecalcified solid tissue histological examination. RESULTS: The border between the material and host's bone was distinct after 4 weeks and blurred after 8 weeks; the density of partial edge of the material was similar to that of radii after 12 weeks. The medullary cavity of bone reopened in group B; the density of most defect area was similar to that of the host bone and there was a few high density shadow in group C; the density of most defect area was higher than that of host bone in group A after 24 weeks. There was no significant difference in radiograph scoring between groups A, B and C after 4 weeks and 8 weeks (P>0.05); the scores of group B and C were higher than that of group A after 12 weeks (P<0.05); and the scores were arranged as follow: group D > group B > group C > group A after 24 weeks (P<0.05). Bone callus grew toward defect area and new bone adhered to the material after 4 weeks and 8 weeks; more new bone formed, and the materials were absorbed and degraded with time. The quantity of bone formation was more in group D than in group B and in group B than in group C and in group C than in group A after 24 weeks (P<0.05). CONCLUSION: PDPB had good osteogenesis in repairing the segmental bone defect, PDCB was inferior to it, both PDPB and PDCB are fit to repair segmental bone defect. Both of them were inferior to autogenous bone.

Dual-Color imaging of nuclear division and mitotic spindle elongation in live cells of Aspergillus nidulans.

We have developed a dual-color imaging system based on cyan fluorescent protein-labeled histone H2A and green fluorescent protein-labeled alpha tubulin to visualize DNA and spindles simultaneously in the same living cell of Aspergillus nidulans. This system allows new details of mitosis and nuclear movement to be revealed.

Accumulation of cytoplasmic dynein and dynactin at microtubule plus ends in Aspergillus nidulans is kinesin dependent.

The mechanism(s) by which microtubule plus-end tracking proteins are targeted is unknown. In the filamentous fungus Aspergillus nidulans, both cytoplasmic dynein and NUDF, the homolog of the LIS1 protein, localize to microtubule plus ends as comet-like structures. Herein, we show that NUDM, the p150 subunit of dynactin, also forms dynamic comet-like structures at microtubule plus ends. By examining proteins tagged with green fluorescent protein in different loss-of-function mutants, we demonstrate that dynactin and cytoplasmic dynein require each other for microtubule plus-end accumulation, and the presence of cytoplasmic dynein is also important for NUDF's plus-end accumulation. Interestingly, deletion of NUDF increases the overall accumulation of dynein and dynactin at plus ends, suggesting that NUDF may facilitate minus-end-directed dynein movement. Finally, we demonstrate that a conventional kinesin, KINA, is required for the microtubule plus-end accumulation of cytoplasmic dynein and dynactin, but not of NUDF.