Mark C. Wagner

Submolecular domains of bovine brain kinesin identified by electron microscopy and monoclonal antibody decoration

Kinesin is a microtubule-activated ATPase thought to transport membrane-bounded organelles along MTs. To illuminate the structural basis for this function, EM was used to locate submolecular domains on bovine brain kinesin. Rotary shadowed kinesin appeared rod-shaped and approximately 80 nm long. One end of each molecule contained a pair of approximately 10 x 9 nm globular domains, while the opposite end was fan-shaped. Monoclonal antibodies against the approximately 124 kd heavy chains of kinesin decorated the globular structures, while those specific for the approximately 64 kd light chains labeled the fan-shaped end. Quick-freeze, deep-etch EM was used to analyze MTs polymerized from tubulin and cross-linked to latex microspheres by kinesin. Microspheres frequently attached to MTs by arm-like structures, 25-30 nm long. The MT attachment sites often appeared as one or two approximately 10 nm globular bulges. Morphologically similar cross-links were observed by quick-freeze, deep-etch EM between organelles and MTs in the neuronal cytoskeleton in vivo. These collective observations suggest that bovine brain kinesin binds to MTs by globular domains that contain the heavy chains, and that the attachment sites for organelles are at the opposite, fan-shaped end of kinesin, where the light chains are located.

Identification of a 120 kd hair-bundle myosin located near stereociliary tips

By adapting to sustained stimuli, hair cells of the internal ear maintain their optimal sensitivity to minute displacements. Biophysical experiments have suggested that adaptation is mediated by a molecular motor, most likely a member of the myosin family. To provide direct evidence for the presence of myosin isozymes in hair bundles, we used photoaffinity labeling with vanadate-trapped uridine and adenine nucleotides to identify proteins of 120, 160, and 230 kd in a preparation of hair bundles purified from the bullfrogs sacculus. The photoaffinity labeling properties of these proteins, particularly the 120 kd protein, resembled those of other well-characterized myosins. A 120 kd hair-bundle protein was also recognized by a monoclonal antibody directed against a vertebrate myosin I isozyme. Immunofluorescence microscopy localized this protein near the beveled top edge of the hair bundle, the site of mechanoelectrical transduction and adaptation.

CaMKII-Mediated Phosphorylation of the Myosin Motor Myo1c Is Required for Insulin-Stimulated GLUT4 Translocation in Adipocytes

The unconventional myosin Myo1c has been implicated in insulin-regulated GLUT4 translocation to the plasma membrane in adipocytes. We show that Myo1c undergoes insulin-dependent phosphorylation at S701. Phosphorylation was accompanied by enhanced 14-3-3 binding and reduced calmodulin binding. Recombinant CaMKII phosphorylated Myo1c in vitro and siRNA knockdown of CaMKIIdelta abolished insulin-dependent Myo1c phosphorylation in vivo. CaMKII activity was increased upon insulin treatment and the CaMKII inhibitors CN21 and KN-62 or the Ca(2+) chelator BAPTA-AM blocked insulin-dependent Myo1c phosphorylation and insulin-stimulated glucose transport in adipocytes. Myo1c ATPase activity was increased after CaMKII phosphorylation in vitro and after insulin stimulation of CHO/IR/IRS-1 cells. Expression of wild-type Myo1c, but not S701A or ATPase dead mutant K111A, rescued the inhibition of GLUT4 translocation by siRNA-mediated Myo1c knockdown. These data suggest that insulin regulates Myo1c function via CaMKII-dependent phosphorylation, and these events play a role in insulin-regulated GLUT4 trafficking in adipocytes likely involving Myo1c motor activity.

Ischemia activates actin depolymerizing factor: role in proximal tubule microvillar actin alterations

Apical membrane of renal proximal tubule cells is extremely sensitive to ischemia, with structural alterations occurring within 5 min. These changes are felt secondary to actin cytoskeletal disruption, yet the mechanism responsible is unknown. Actin depolymerizing factor (ADF), a 19-kDa actin-binding protein, has recently been shown to play an important role in regulation of actin filament dynamics. Because ADF is known to mediate pH-dependent F-actin binding, depolymerization, and severing, and because ADF activation occurs by dephosphorylation, we questioned whether ADF played a role in microvilli microfilament disruption during ischemia. To test our hypothesis, we induced renal ischemia in the rat with the clamp model. Initial immunofluorescence and Western blot studies on cortical tissue documented the presence of ADF in proximal tubule cells. Under physiological conditions, ADF was distributed homogeneously throughout the cytoplasm, primarily in the Triton X-100-soluble fraction, and both phosphorylated (pADF) and nonphosphorylated forms were identified. During ischemia, marked alterations occurred. Intraluminal vesicle/bleb structures contained extremely high concentrations of ADF along with G-actin, but not F-actin. Western blot showed a rapidly occurring duration-dependent dephosphorylation of ADF. At 0-30 min of ischemia, total ADF levels were unchanged, whereas pADF decreased significantly to 72% and 19% of control levels, at 5 and 15 min, respectively. Urine collected under physiological conditions did not contain ADF or actin, whereas urine collected after 30 min of ischemia contained both ADF and actin. Reperfusion was associated with normalization of cellular pADF levels, pADF intracellular distribution, and repair of apical microvilli. These data suggest that activation of ADF during ischemia via dephosphorylation is, in part, responsible for apical actin disruption resulting in microvillar destruction and formation of intraluminal vesicles.

Role of the actin cytoskeleton in ischemia-induced cell injury and repair.

This paper reviews the role of the actin cytoskeleton in the establishment and maintenance of surface membrane structure and function in all epithelial cells. It describes in detail certain interactions between the actin cytoskeleton and the surface membrane. Recent studies show that ischemia and/or ATP depletion will rapidly disrupt the actin cytoskeleton, an important event in ischemia-induced cell injury. Finally, the review examines specific functional and structural interactions between the actin cytoskeleton and the surface membrane.

Multiple Factors Influence Glomerular Albumin Permeability in Rats

Different laboratories recently reported incongruous results describing the quantification of albumin filtration using two-photon microscopy. We investigated the factors that influence the glomerular sieving coefficient for albumin (GSC(A)) in an effort to explain these discordant reports and to develop standard operating procedures for determining GSC(A). Multiple factors influenced GSC(A), including the kidney depth of image acquisition (10-20 μm was appropriate), the selection of fluorophore (probes emitting longer wavelengths were superior), the selection of plasma regions for fluorescence measurements, the size and molecular dispersion characteristics of dextran polymers if used, dietary status, and the genetic strain of rat. Fasting reduced the GSC(A) in Simonsen Munich Wistar rats from 0.035±0.005 to 0.016±0.004 (P<0.01). Frömter Munich Wistar rats had a much lower GSC(A) in both the fed and the fasted states. Finally, we documented extensive albumin transcytosis with vesicular and tubular delivery to and fusion with the basolateral membrane in S1 proximal tubule cells. In summary, these results help explain the previously conflicting microscopy and micropuncture data describing albumin filtration and highlight the dynamic nature of glomerular albumin permeability.

Motor Protein Myo1c Is a Podocyte Protein That Facilitates the Transport of Slit Diaphragm Protein Neph1 to the Podocyte Membrane

ABSTRACT The podocyte proteins Neph1 and nephrin organize a signaling complex at the podocyte cell membrane that forms the structural framework for a functional glomerular filtration barrier. Mechanisms regulating the movement of these proteins to and from the membrane are currently unknown. This study identifies a novel interaction between Neph1 and the motor protein Myo1c, where Myo1c plays an active role in targeting Neph1 to the podocyte cell membrane. Using in vivo and in vitro experiments, we provide data supporting a direct interaction between Neph1 and Myo1c which is dynamic and actin dependent. Unlike wild-type Myo1c, the membrane localization of Neph1 was significantly reduced in podocytes expressing dominant negative Myo1c. In addition, Neph1 failed to localize at the podocyte cell membrane and cell junctions in Myo1c-depleted podocytes. We further demonstrate that similarly to Neph1, Myo1c also binds nephrin and reduces its localization at the podocyte cell membrane. A functional analysis of Myo1c knockdown cells showed defects in cell migration, as determined by a wound assay. In addition, the ability to form tight junctions was impaired in Myo1c knockdown cells, as determined by transepithelial electric resistance (TER) and bovine serum albumin (BSA) permeability assays. These results identify a novel Myo1c-dependent molecular mechanism that mediates the dynamic organization of Neph1 and nephrin at the slit diaphragm and is critical for podocyte function.

DNA mismatch repair (MMR)-dependent 5-fluorouracil cytotoxicity and the potential for new therapeutic targets

The metabolism and efficacy of 5‐fluorouracil (FUra) and other fluorinated pyrimidine (FP) derivatives have been intensively investigated for over fifty years. FUra and its antimetabolites can be incorporated at RNA‐ and DNA‐levels, with RNA level incorporation provoking toxic responses in human normal tissue, and DNA‐level antimetabolite formation and incorporation believed primarily responsible for tumour‐selective responses. Attempts to direct FUra into DNA‐level antimetabolites, based on mechanism‐of‐action studies, have led to gradual improvements in tumour therapy. These include the use of leukovorin to stabilize the inhibitory thymidylate synthase‐5‐fluoro‐2′‐deoxyuridine 5′ monophoshate (FdUMP)‐5,10‐methylene tetrahydrofolate (5,10‐CH2FH4) trimeric complex. FUra incorporated into DNA also contributes to antitumour activity in preclinical and clinical studies. This review examines our current state of knowledge regarding the mechanistic aspects of FUra:Gua lesion detection by DNA mismatch repair (MMR) machinery that ultimately results in lethality. MMR‐dependent direct cell death signalling or futile cycle responses will be discussed. As 10–30% of sporadic colon and endometrial tumours display MMR defects as a result of human MutL homologue‐1 (hMLH1) promoter hypermethylation, we discuss the use and manipulation of the hypomethylating agent, 5‐fluorodeoxycytidine (FdCyd), and our ability to manipulate its metabolism using the cytidine or deoxycytidylate (dCMP) deaminase inhibitors, tetrahydrouridine or deoxytetrahydrouridine, respectively, as a method for re‐expression of hMLH1 and re‐sensitization of tumours to FP therapy.

Renal epithelial polarity in health and disease

Abstract Epithelial cells mediate the unidirectional movement of selective compounds from one biological compartment to another. This is accomplished by having biochemically, structurally, and functionally distinctive apical and basolateral surface membrane domains separated by the cells’ junctional complex. Derangement of this highly ordered situation can result in cell injury, dysfunction, and even death. For renal epithelial cells, both ischemia and polycystic kidney disease are known to result in a loss of surface membrane polarity. In both disease processes, this in turn plays an important role in cell and organ dysfunction.

Purification of kinesin from bovine brain and assay of microtubule-stimulated ATPase activity

The protocols described here have proved to be an effective method for preparation of kinesin suitable for biochemical, biophysical, and immunological analyses. Beginning with a 1.2-liter cytosolic extract of bovine brain containing approximately 24 g of protein, 2 mg of approximately 95% pure kinesin can be obtained within 2 days. There are four major enrichment steps, as summarized in Fig. 6 and Table I. Based on quantitative SDS-PAGE, we estimate that these steps result in a purification of more than 300-fold. The ATPase activity in the presence of microtubules is substantial, and the kinetic properties are consistent with cellular levels of ATP (Km approximately 0.2 mM) and microtubules (apparent Km for activation approximately 1.9 microM) in the axon. Minor modifications should allow the procedure to be enlarged or reduced in scale, or adapted to the brains of other vertebrate species. The availability of such procedures will greatly facilitate future studies of the cell and molecular biology of kinesin.