Kevin W.-H. Lo

The 8-kDa Dynein Light Chain Binds to Its Targets via a Conserved (K/R)XTQT Motif

Cytoplasmic dynein is a large, multisubunit molecular motor that translocates cargoes toward the minus ends of microtubules. Proper functioning of the dynein motor requires precise assembly of its various subunits. Using purified recombinant proteins, we show that the highly conserved 8-kDa light chain (DLC8) binds to the intermediate chain of the dynein complex. The DLC8-binding region was mapped to a highly conserved 10-residue fragment (amino acid sequence SYSKETQTPL) C-terminal to the second alternative splicing site of dynein intermediate chain. Yeast two-hybrid screening using DLC8 as bait identified numerous additional DLC8-binding proteins. Biochemical and mutational analysis of selected DLC8-binding proteins revealed that DLC8 binds to a consensus sequence containing a (K/R)XTQT motif. The (K/R)XTQT motif interacts with the common target-accepting grooves of DLC8 dimer. The role of each conserved amino acid residue in this pentapeptide motif in supporting complex formation with DLC8 was systematically studied using site-directed mutagenesis.

Studies of bone morphogenetic protein-based surgical repair.

Over the past several decades, recombinant human bone morphogenetic proteins (rhBMPs) have been the most extensively studied and widely used osteoinductive agents for clinical bone repair. Since rhBMP-2 and rhBMP-7 were cleared by the U.S. Food and Drug Administration for certain clinical uses, millions of patients worldwide have been treated with rhBMPs for various musculoskeletal disorders. Current clinical applications include treatment of long bone fracture non-unions, spinal surgeries, and oral maxillofacial surgeries. Considering the growing number of recent publications related to clincal research of rhBMPs, there exists enormous promise for these proteins to be used in bone regenerative medicine. The authors take this opportunity to review the rhBMP literature paying specific attention to the current applications of rhBMPs in bone repair and spine surgery. The prospective future of rhBMPs delivered in combination with tissue engineered scaffolds is also reviewed.

Structure of Tctex-1 and its interaction with cytoplasmic dynein intermediate chain.

The minus-ended microtubule motor cytoplasmic dynein contains a number of low molecular weight light chains including the 14-kDa Tctex-1. The assembly of Tctex-1 in the dynein complex and its function are largely unknown. Using partially deuterated, 15N,13C-labeled protein samples and transverse relaxation-optimized NMR spectroscopic techniques, the secondary structure and overall topology of Tctex-1 were determined based on the backbone nuclear Overhauser effect pattern and the chemical shift values of the protein. The data showed that Tctex-1 adopts a structure remarkably similar to that of the 8-kDa light chain of the motor complex (DLC8), although the two light chains share no amino acid sequence homology. We further demonstrated that Tctex-1 binds directly to the intermediate chain (DIC) of dynein. The Tctex-1 binding site on DIC was mapped to a 19-residue fragment immediately following the second alternative splicing site of DIC. Titration of Tctex-1 with a peptide derived from DIC, which contains a consensus sequence R/KR/KXXR/K found in various Tctex-1 target proteins, indicated that Tctex-1 binds to its targets in a manner similar to that of DLC8. The experimental results presented in this study suggest that Tctex-1 is likely to be a specific cargo adaptor for the dynein motor complex.

A neuron-specific cytoplasmic dynein isoform preferentially transports TrkB signaling endosomes

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.

Trk Activation of the ERK1/2 Kinase Pathway Stimulates Intermediate Chain Phosphorylation and Recruits Cytoplasmic Dynein to Signaling Endosomes for Retrograde Axonal Transport

The retrograde transport of Trk-containing endosomes from the axon to the cell body by cytoplasmic dynein is necessary for axonal and neuronal survival. We investigated the recruitment of dynein to signaling endosomes in rat embryonic neurons and PC12 cells. We identified a novel phosphoserine on the dynein intermediate chains (ICs), and we observed a time-dependent neurotrophin-stimulated increase in intermediate chain phosphorylation on this site in both cell types. Pharmacological studies, overexpression of constitutively active MAP kinase kinase, and an in vitro assay with recombinant proteins demonstrated that the intermediate chains are phosphorylated by the MAP kinase ERK1/2, extracellular signal-regulated kinase, a major downstream effector of Trk. Live cell imaging with fluorescently tagged IC mutants demonstrated that the dephosphomimic mutants had significantly reduced colocalization with Trk and Rab7, but not a mitochondrial marker. The phosphorylated intermediate chains were enriched on immunoaffinity-purified Trk-containing organelles. Inhibition of ERK reduced the amount of phospho-IC and the total amount of dynein that copurified with the signaling endosomes. In addition, inhibition of ERK1/2 reduced the motility of Rab7- and TrkB-containing endosomes and the extent of their colocalization with dynein in axons. NGF-dependent survival of sympathetic neurons was significantly reduced by the overexpression of the dephosphomimic mutant IC-1B-S80A, but not WT IC-1B, further demonstrating the functional significance of phosphorylation on this site. These results demonstrate that neurotrophin binding to Trk initiates the recruitment of cytoplasmic dynein to signaling endosomes through ERK1/2 phosphorylation of intermediate chains for their subsequent retrograde transport in axons.

Delivery of small molecules for bone regenerative engineering: preclinical studies and potential clinical applications.

Stimulation of bone regeneration using growth factors is a promising approach for musculoskeletal regenerative engineering. However, common limitations with protein growth factors, such as high manufacturing costs, protein instability, contamination issues, and unwanted immunogenic responses of the host reduce potential clinical applications. New strategies for bone regeneration that involve inexpensive and stable small molecules can obviate these problems and have a significant impact on the treatment of skeletal injury and diseases. Over the past decade, a large number of small molecules with the potential of regenerating skeletal tissue have been reported in the literature. Here, we review this literature, paying specific attention to the prospects for small molecule-based bone-regenerative engineering. We also review the preclinical study of small molecules associated with bone regeneration.

The role of small molecules in musculoskeletal regeneration

The uses of bone morphogenetic proteins and parathyroid hormone therapeutics are fraught with several fundamental problems, such as cost, protein stability, immunogenicity, contamination and supraphysiological dosage. These downsides may effectively limit their more universal use. Therefore, there is a clear need for alternative forms of biofactors to obviate the drawbacks of protein-based inductive factors for bone repair and regeneration. Our group has studied small molecules with the capacity to regulate osteoblast differentiation and mineralization because their inherent physical properties minimize limitations observed in protein growth factors. For instance, in general, small molecule inducers are usually more stable, highly soluble, nonimmunogenic, more affordable and require lower dosages. Small molecules with the ability to induce osteoblastic differentiation may represent the next generation of bone regenerative medicine. This review describes efforts to develop small molecule-based biofactors for induction, paying specific attention to their novel roles in bone regeneration.

Small-molecule based musculoskeletal regenerative engineering.

Clinicians and scientists working in the field of regenerative engineering are actively investigating a wide range of methods to promote musculoskeletal tissue regeneration. Small-molecule-mediated tissue regeneration is emerging as a promising strategy for regenerating various musculoskeletal tissues and a large number of small-molecule compounds have been recently discovered as potential bioactive molecules for musculoskeletal tissue repair and regeneration. In this review, we summarize the recent literature encompassing the past 4 years in the area of small bioactive molecules for promoting repair and regeneration of various musculoskeletal tissues including bone, muscle, cartilage, tendon, and nerve.

The small molecule PKA-specific cyclic AMP analogue as an inducer of osteoblast-like cells differentiation and mineralization.

Osteoblastic differentiation is an important landmark for bone formation, bone repair and regeneration; however, it is a very complex process controlled by different signalling mechanisms. Several groups have reported that the cyclic adenosine monophosphate (cAMP) signalling system is responsible for regulating osteoblast cell differentiation. Nonetheless, to date, the principle role of the cAMP molecules related to this process remains controversial. Moreover, the underlying cAMP‐dependent signalling cascade governing the osteoblastic differentiation has not been clarified. In this study we investigated the roles of the cAMP‐dependent protein kinase A (PKA) signalling in proliferation, differentiation and mineralization of osteoblast‐like MC3T3‐E1 cells, using the PKA‐specific small molecule cAMP analogue, 6‐Bnz‐cAMP, at 100 µM. Alkaline phosphatase (ALP) activity, runt transcription factor 2 (Runx2), osteopontin (OPN) and osteocalcin (OCN) protein expressions were used as osteoblast‐specific markers to demonstrate osteoblastic differentiation. Further, calcium measurement of the extracellular matrix was employed as the hallmark of matrix mineralization or calcification. We report here that activation of PKA by the small molecule 6‐Bnz‐cAMP induces osteoblastic differentiation and matrix mineralization of osteoblast‐like MC3T3‐E1 cells. Moreover, 6‐Bnz‐cAMP does not induce cytotoxicity to the cells, as revealed by our cell proliferation studies. Therefore, based on these findings, we propose that the PKA‐specific small molecule 6‐Bnz‐cAMP may serve as a novel bone‐inducing growth factor for repairing and regenerating bone tissues during bone‐regenerative engineering. Copyright

The DYNLT3 Light Chain Directly Links Cytoplasmic Dynein to a Spindle Checkpoint Protein, Bub3

Cytoplasmic dynein is the motor protein responsible for the intracellular transport of various organelles and other cargoes toward microtubule minus ends. However, it remains to be determined how dynein is regulated to accomplish its varied roles. The dynein complex contains six subunits, including three classes of light chains. The two isoforms of the DYNLT (Tctex1) family of light chains, DYNLT1 and DYNLT3, have been proposed to link dynein to specific cargoes. However, no specific binding partner had been found for the DYNLT3 light chain. We find that DYNLT3 binds to Bub3, a spindle checkpoint protein. Bub3 binds exclusively to DYNLT3 and not to the other dynein light chains. Glutathione S-transferase pull-down and co-immunoprecipitation assays demonstrate that Bub3 interacts with the cytoplasmic dynein complex. DYNLT3 is present on kinetochores at prometaphase, but not later mitotic stages, demonstrating that this dynein light chain, like Bub3 and other checkpoint proteins, is depleted from the kinetochore during chromosome alignment. Knockdown of DYNLT3 with small interference RNA increases the mitotic index, in particular, the number of cells in prophase/prometaphase. These results demonstrate that dynein binds directly to a component of the spindle checkpoint complex through the DYNLT3 light chain. Thus, DYNLT3 contributes to dynein cargo binding specificity. These data also suggest that the subpopulation of dynein, containing the DYNLT3 light chain, may be important for chromosome congression, in addition to having a role in the transport of checkpoint proteins from the kinetochore to the spindle pole.