Farida Safadi

The Kinesin-like Calmodulin Binding Protein Is Differentially Involved in Cell Division

The kinesin-like calmodulin (CaM) binding protein (KCBP), a minus end–directed microtubule motor protein unique to plants, has been implicated in cell division. KCBP is negatively regulated by Ca2+ and CaM, and antibodies raised against the CaM binding region inhibit CaM binding to KCBP in vitro; therefore, these antibodies can be used to activate KCBP constitutively. Injection of these antibodies into Tradescantia virginiana stamen hair cells during late prophase induces breakdown of the nuclear envelope within 2 to 10 min and leads the cell into prometaphase. However, mitosis is arrested, and the cell does not progress into anaphase. Injection of antibodies later during cell division has no effect on anaphase transition but causes aberrant phragmoplast formation and delays the completion of cytokinesis by ∼15 min. These effects are achieved without any apparent degradation of the microtubule cytoskeleton. We propose that during nuclear envelope breakdown and anaphase, activated KCBP promotes the formation of a converging bipolar spindle by sliding and bundling microtubules. During metaphase and telophase, we suggest that its activity is downregulated.

Interaction of a Kinesin-like Protein with Calmodulin Isoforms from Arabidopsis*

In Arabidopsis and other plants there are multiple calmodulin isoforms. However, the role of these isoforms in regulating the activity of target proteins is obscure. Here, we analyzed the interaction between a kinesin-like calmodulin-binding motor protein (Reddy, A. S. N., Safadi, F., Narasimhulu, S. B., Golovkin, M., and Hu, X. (1996) J. Biol. Chem. 271, 7052–7060) and three calmodulin isoforms (calmodulin-2, -4, and -6) from Arabidopsis using different approaches. Gel mobility and fluorescence shift assays revealed that the motor binds to all calmodulin isoforms in a calcium-dependent manner. Furthermore, all calmodulin isoforms were able to activate bovine calcium/calmodulin-dependent phosphodiesterase. However, the concentration of calmodulin-2 required for half-maximal activation of phosphodiesterase is 2- and 6-fold lower compared with calmodulin-4 and -6, respectively. The dissociation constants of the motor to calmodulin-2, -4, and -6 are 12.8, 27.0, and 27.8 nm, respectively, indicating that calmodulin-2 has 2-fold higher affinity for the motor than calmodulin-4 and -6. Similar results were obtained using another assay that involves the binding of35S-labeled calmodulin isoforms to the motor. The binding saturation curves of the motor with calmodulin isoforms have confirmed that calmodulin-2 has 2-fold higher affinity to the motor. However, the affinity of calmodulin-4 and -6 isoforms for the motor was about the same. Based on these studies, we conclude that all calmodulin isoforms bind to the motor protein but with different affinities.

A pollen-specific novel calmodulin-binding protein with tetratricopeptide repeats

Calcium is essential for pollen germination and pollen tube growth. A large body of information has established a link between elevation of cytosolic Ca2+ at the pollen tube tip and its growth. Since the action of Ca2+ is primarily mediated by Ca2+-binding proteins such as calmodulin (CaM), identification of CaM-binding proteins in pollen should provide insights into the mechanisms by which Ca2+regulates pollen germination and tube growth. In this study, a CaM-binding protein from maize pollen (maizepollen calmodulin-bindingprotein, MPCBP) was isolated in a protein-protein interaction-based screening using 35S-labeled CaM as a probe. MPCBP has a molecular mass of about 72 kDa and contains three tetratricopeptide repeats (TPR) suggesting that it is a member of the TPR family of proteins. MPCBP protein shares a high sequence identity with two hypothetical TPR-containing proteins fromArabidopsis. Using gel overlay assays and CaM-Sepharose binding, we show that the bacterially expressed MPCBP binds to bovine CaM and three CaM isoforms from Arabidopsis in a Ca2+-dependent manner. To map the CaM-binding domain several truncated versions of the MPCBP were expressed in bacteria and tested for their ability to bind CaM. Based on these studies, the CaM-binding domain was mapped to an 18-amino acid stretch between the first and second TPR regions. Gel and fluorescence shift assays performed with CaM and a CaM-binding synthetic peptide further confirmed MPCBP binding to CaM. Western, Northern, and reverse transcriptase-polymerase chain reaction analysis have shown that MPCBP expression is specific to pollen. MPCBP was detected in both soluble and microsomal proteins. Immunoblots showed the presence of MPCBP in mature and germinating pollen. Pollen-specific expression of MPCBP, its CaM-binding properties, and the presence of TPR motifs suggest a role for this protein in Ca2+-regulated events during pollen germination and growth.

Studies on stomatal function, epicuticular wax and stem-root transition region of polyethylene glycol-treated and nontreated in vitro grape plantlets

SummaryScanning electron microscopy, light microscopy, and gravimetric analysis was used to evaluate stomatal function, epicuticular wax, and the stem-root transition region of grape (Vitis sp. ‘Valiant’) plantlets grownin vitro, polyethylene glycoltreatedin vitro, and greenhouse-grown plants. Scanning electron microscopic studies of leaf surfaces ofin vitro-grown plants showed widely open stomata as compared to leaf stomata of polyethylene glycol-treatedin vitro-cultured and greenhouse-grown plants. Ultrastructurally, leaf epicuticular wax ofin vitro plants was less dense than in their polyethylene-treated and greenhouse counterparts. Quantitatively,in vitro-grown plants had reduced epicuticular was as compared to polyethylene glycol-treated and greenhouse-grown plants. Light microscopic studies showed no obvious differences in the vascular connections in the stem-root transition region ofin vitro-cultured, polyethylene glycol-treatedin vitro-cultured, and greenhouse-grown plants. It is therefore likely that the rapid wilting and desiccation observed after transplantingin vitro grape plantlets is due to their defective stomatal function and reduced epicuticular wax and may not be due to poor water transport associated with vascular connection.