Douglas A. Weidner

Efficient delivery of small interfering RNA to plant cells by a nanosecond pulsed laser-induced stress wave for posttranscriptional gene silencing

Small interfering RNA (siRNA) induced posttranscriptional gene silencing (PTGS) has been an efficient method for genetic and molecular analysis of certain developmental and physiological processes and represented a potential strategy for both controlling virus replication and developing therapeutic products. However, there are limitations for the methods currently used to deliver siRNA into cells. We report here, to our knowledge, the first efficient delivery of siRNA to plant cells by a nanosecond pulsed laser-induced stress wave (LISW) for posttranscriptional gene silencing. Using LISW, we are able to silence gene expression in cell cultures of three different plant species rice (Oryza sativa L.), cotton (Gossypium hirsutum L.), and slash pine (Pinus elliottii Engelm.). Gene silencing induced by siRNA has been confirmed by northern blot, laser scanning microscopy, and siRNA analysis. These data suggested that LISW-mediated siRNA delivery can be a reliable and effective method for inducing PTGS in cultured cells.

Role of benzo[α]pyrene in generation of clustered DNA damage in human breast tissue

Complex DNA damage may manifest in double-strand breaks (DSBs) and non-DSB, bistranded, oxidatively induced clustered DNA lesions (OCDLs). Although the carcinogen benzo[alpha]pyrene (B[alpha]P) has been shown to induce chromosomal aberrations and transformation of mammary cells, it is not known whether this compound engenders clustered DNA damage. Normal primary breast tissue-derived cells were treated with B[alpha]P, and the levels of DNA lesions, chromosomal aberrations, total antioxidant capacity (TAC), and reactive oxygen species (ROS) were determined. DNA from cells treated with 2 and 8 microM B[alpha]P exhibited increases of 3- and 4-fold in APE1 (p<0.001), 11- and 19-fold in Endo III (p<0.001), and 8- and 15-fold in hOGG1 (p<0.001) OCDLs, respectively, compared to the 0 microM B[alpha]P-treated (control) group. Mammary cells treated with 8 microM B[alpha]P produced 0.12 aberrations per cell (p<0.05) and there was a strong positive correlation (r=0.91) between the levels of OCDLs and those of chromosomal aberrations. Finally, TAC was decreased by 25% (p<0.02), whereas ROS production increased by 2-fold (p<0.02) in cells treated with 8 microM B[alpha]P compared to the control group. In conclusion, oxidatively induced clustered DNA damage mediated through differential expression of APE1, reduced TAC, and increased ROS may play a significant role in the chemically induced transformation of normal primary mammary cells.

Glycan Structures Contain Information for the Spatial Arrangement of Glycoproteins in the Plasma Membrane

Glycoconjugates at the cell surface are crucial for cells to communicate with each other and the extracellular microenvironment. While it is generally accepted that glycans are vectorial biopolymers, their information content is unclear. This report provides evidence that distinct N-glycan structures influence the spatial arrangement of two integral membrane glycoproteins, Kv3.1 and E-cadherin, at the adherent membrane which in turn alter cellular properties. Distinct N-glycan structures were generated by heterologous expression of these glycoproteins in parental and glycosylation mutant Chinese hamster ovary cell lines. Unlike the N-linked glycans, the O-linked glycans of the mutant cell lines are similar to those of the parental cell line. Western and lectin blots of total membranes and GFP immunopurified samples, combined with glycosidase digestion reactions, were employed to verify the glycoproteins had predominantly complex, oligomannose, and bisecting type N-glycans from Pro-5, Lec1, and Lec10B cell lines, respectively. Based on total internal reflection fluorescence and differential interference contrast microscopy techniques, and cellular assays of live parental and glycosylation mutant CHO cells, we propose that glycoproteins with complex, oligomannose or bisecting type N-glycans relay information for localization of glycoproteins to various regions of the plasma membrane in both a glycan-specific and protein-specific manner, and furthermore cell-cell interactions are required for deciphering much of this information. These distinct spatial arrangements also impact cell adhesion and migration. Our findings provide direct evidence that N-glycan structures of glycoproteins contribute significantly to the information content of cells.

N-Linked glycan site occupancy impacts the distribution of a potassium channel in the cell body and outgrowths of neuronal-derived cells

BACKGROUND Vacancy of occupied N-glycosylation sites of glycoproteins is quite disruptive to a multicellular organism, as underlined by congenital disorders of glycosylation. Since a neuronal component is typically associated with this disease, we evaluated the impact of N-glycosylation processing of a neuronal voltage gated potassium channel, Kv3.1b, expressed in a neuronal-derived cell line, B35 neuroblastoma cells. METHODS Total internal reflection fluorescence and differential interference contrast microscopy measurements of live B35 cells expressing wild type and glycosylation mutant Kv3.1b proteins were used to evaluate the distribution of the various forms of the Kv3.1b protein in the cell body and outgrowths. Cell adhesion assays were also employed. RESULTS Microscopy images revealed that occupancy of both N-glycosylation sites of Kv3.1b had relatively similar amounts of Kv3.1b in the outgrowth and cell body while vacancy of one or both sites led to increased accumulation of Kv3.1b in the cell body. Further both the fully glycosylated and partially glycosylated N229Q Kv3.1b proteins formed higher density particles in outgrowths compared to cell body. Cellular assays demonstrated that the distinct spatial arrangements altered cell adhesion properties. CONCLUSIONS Our findings provide direct evidence that occupancy of the N-glycosylation sites of Kv3.1b contributes significantly to its lateral heterogeneity in membranes of neuronal-derived cells, and in turn alters cellular properties. GENERAL SIGNIFICANCE Our study demonstrates that N-glycans of Kv3.1b contain information regarding the association, clustering, and distribution of Kv3.1b in the cell membrane, and furthermore that decreased occupancy caused by congenital disorders of glycosylation may alter the biological activity of Kv3.1b.

Soluble guanylyl cyclase-activated cyclic GMP-dependent protein kinase inhibits arterial smooth muscle cell migration independent of VASP-serine 239 phosphorylation

Coronary artery disease (CAD) accounts for over half of all cardiovascular disease-related deaths. Uncontrolled arterial smooth muscle (ASM) cell migration is a major component of CAD pathogenesis and efforts aimed at attenuating its progression are clinically essential. Cyclic nucleotide signaling has long been studied for its growth-mitigating properties in the setting of CAD and other vascular disorders. Heme-containing soluble guanylyl cyclase (sGC) synthesizes cyclic guanosine monophosphate (cGMP) and maintains vascular homeostasis predominantly through cGMP-dependent protein kinase (PKG) signaling. Considering that reactive oxygen species (ROS) can interfere with appropriate sGC signaling by oxidizing the cyclase heme moiety and so are associated with several CVD pathologies, the current study was designed to test the hypothesis that heme-independent sGC activation by BAY 60-2770 (BAY60) maintains cGMP levels despite heme oxidation and inhibits ASM cell migration through phosphorylation of the PKG target and actin-binding vasodilator-stimulated phosphoprotein (VASP). First, using the heme oxidant ODQ, cGMP content was potentiated in the presence of BAY60. Using a rat model of arterial growth, BAY60 significantly reduced neointima formation and luminal narrowing compared to vehicle (VEH)-treated controls. In rat ASM cells BAY60 significantly attenuated cell migration, reduced G:F actin, and increased PKG activity and VASP Ser239 phosphorylation (pVASP·S239) compared to VEH controls. Site-directed mutagenesis was then used to generate overexpressing full-length wild type VASP (FL-VASP/WT), VASP Ser239 phosphorylation-mimetic (FL-VASP/239D) and VASP Ser239 phosphorylation-resistant (FL-VASP/239A) ASM cell mutants. Surprisingly, FL-VASP/239D negated the inhibitory effects of FL-VASP/WT and FL-VASP/239A cells on migration. Furthermore, when FL-VASP mutants were treated with BAY60, only the FL-VASP/239D group showed reduced migration compared to its VEH controls. Intriguingly, FL-VASP/239D abrogated the stimulatory effects of FL-VASP/WT and FL-VASP/239A cells on PKG activity. In turn, pharmacologic blockade of PKG in the presence of BAY60 reversed the inhibitory effect of BAY60 on naïve ASM cell migration. Taken together, we demonstrate for the first time that BAY60 inhibits ASM cell migration through cGMP/PKG/VASP signaling yet through mechanisms independent of pVASP·S239 and that FL-VASP overexpression regulates PKG activity in rat ASM cells. These findings implicate BAY60 as a potential pharmacotherapeutic agent against aberrant ASM growth disorders such as CAD and also establish a unique mechanism through which VASP controls PKG activity.

Cell surface N‐glycans influence the level of functional E‐cadherin at the cell–cell border

E‐cadherin is crucial for adhesion of cells to each other and thereby development and maintenance of tissue. While it is has been established that N‐glycans inside the cell impact the level of E‐cadherin at the cell surface of epithelial‐derived cells, it is unclear whether N‐glycans outside the cell control the clustering of E‐cadherin at the cell–cell border. Here, we demonstrate reduction of N‐glycans at the cell surface weakened the recruitment and retention of E‐cadherin at the cell–cell border, and consequently reduced the strength of cell–cell interactions. We conclude that N‐glycans at the cell surface are tightly linked to the placement of E‐cadherin at the cell–cell border and thereby control E‐cadherin mediated cell–cell adhesion.

Predominant Expression of Hybrid N-Glycans Has Distinct Cellular Roles Relative to Complex and Oligomannose N-Glycans.

Glycosylation modulates growth, maintenance, and stress signaling processes. Consequently, altered N-glycosylation is associated with reduced fitness and disease. Therefore, expanding our understanding of N-glycans in altering biological processes is of utmost interest. Herein, clustered regularly interspaced short palindromic repeats/caspase9 (CRISPR/Cas9) technology was employed to engineer a glycosylation mutant Chinese Hamster Ovary (CHO) cell line, K16, which expresses predominantly hybrid type N-glycans. This newly engineered cell line enabled us to compare N-glycan effects on cellular properties of hybrid type N-glycans, to the well-established Pro−5 and Lec1 cell lines, which express complex and oligomannose types of N-glycans, respectively. Lectin binding studies revealed the predominant N-glycan expressed in K16 is hybrid type. Cell dissociation and migration assays demonstrated the greatest strength of cell–cell adhesion and fastest migratory rates for oligomannose N-glycans, and these properties decreased as oligomannose type were converted to hybrid type, and further decreased upon conversion to complex type. Next, we examined the roles of three general types of N-glycans on ectopic expression of E-cadherin, a cell–cell adhesion protein. Microscopy revealed more functional E-cadherin at the cell–cell border when N-glycans were oligomannose and these levels decreased as the oligomannose N-glycans were processed to hybrid and then to complex. Thus, we provide evidence that all three general types of N-glycans impact plasma membrane architecture and cellular properties.

Complex N-Glycans Influence the Spatial Arrangement of Voltage Gated Potassium Channels in Membranes of Neuronal-Derived Cells

The intrinsic electrical properties of a neuron depend on expression of voltage gated potassium (Kv) channel isoforms, as well as their distribution and density in the plasma membrane. Recently, we showed that N-glycosylation site occupancy of Kv3.1b modulated its placement in the cell body and neurites of a neuronal-derived cell line, B35 neuroblastoma cells. To extrapolate this mechanism to other N-glycosylated Kv channels, we evaluated the impact of N-glycosylation occupancy of Kv3.1a and Kv1.1 channels. Western blots revealed that wild type Kv3.1a and Kv1.1 α-subunits had complex and oligomannose N-glycans, respectively, and that abolishment of the N-glycosylation site(s) generated Kv proteins without N-glycans. Total internal reflection fluorescence microscopy images revealed that N-glycans of Kv3.1a contributed to its placement in the cell membrane while N-glycans had no effect on the distribution of Kv1.1. Based on particle analysis of EGFP-Kv proteins in the adhered membrane, glycosylated forms of Kv3.1a, Kv1.1, and Kv3.1b had differences in the number, size or density of Kv protein clusters in the cell membrane of neurites and cell body of B35 cells. Differences were also observed between the unglycosylated forms of the Kv proteins. Cell dissociation assays revealed that cell-cell adhesion was increased by the presence of complex N-glycans of Kv3.1a, like Kv3.1b, whereas cell adhesion was similar in the oligomannose and unglycosylated Kv1.1 subunit containing B35 cells. Our findings provide direct evidence that N-glycans of Kv3.1 splice variants contribute to the placement of these glycoproteins in the plasma membrane of neuronal-derived cells while those of Kv1.1 were absent. Further when the cell membrane distribution of the Kv channel was modified by N-glycans then the cell-cell adhesion properties were altered. Our study demonstrates that N-glycosylation of Kv3.1a, like Kv3.1b, provides a mechanism for the distribution of these proteins to the cell body and outgrowths and thereby can generate different voltage-dependent conductances in these membranes.

Flow cytometry and fluorescence in situ hybridization to detect residual neuroblastoma cells in bone marrow

In patients with neuroblastoma morphological assessment of BM for residual NB cells is not precise, particularly when the number of tumor cells is small.

Membrane Distribution and Activity of a Neuronal Voltage-Gated K+ Channel is Modified by Replacement of Complex Type N-Glycans with Hybrid Type

Abnormal modifications in N-glycosylation processing are commonly associated with neurological disorders, although the impact of specific N-glycans on neuronal excitability is unknown. By replacement of complex types of N-glycans with hybrid types in neuroblastoma cells, we provide the first study that addresses how distinct N-glycan types impact neuronal excitability. Using CRISPR/Cas9 technology, NB_1, a clonal cell line derived from rat neuroblastoma cells (NB), was modified to create an N-glycosylation mutant cell line, NB_1 (-Mgat2), which expresses predominantly hybrid type N-glycans. Western and lectin blotting, flow cytometry, TIRF and DIC microscopy, and patch clamp studies were conducted. Lectin binding revealed the predominant type of N-glycans expressed in NB_1 (-Mgat2) is hybrid while those of NB and NB_1 are complex. Kv3.1 b-expressing cells with complex N-glycans localized more glycosylated Kv3.1b to the neurites than cells with hybrid N-glycans. Further the absence of N-glycan attachment to Kv3.1b was critical for sub-plasma distribution of Kv3.1b to neurites in primary adult mammalian neurons, along with NB cells. Replacement of complex type N-glycans with hybrid type hindered the opening and closing rates of outward ionic currents of Kv3.1 b-expressing NB cells. The lacks of N-glycan attachment hindered the rates even more but were not significantly different between the NB cell lines. Taken together, our evidence supports N-glycosylation impacts the sub-plasma membrane localization and activity of Kv3.1 b-containing channels. We propose that N-glycosylation processing of Kv3.1 b-containing channels contributes to neuronal excitability, and abnormal modifications in N-glycosylation processing of Kv3.1b could contribute to neurological diseases.