GATA2 regulates blood/lymph separation in a platelet-dependent and lymphovenous valve-independent manner.

INTRODUCTION: Lymphatic vessels collect interstitial fluid, immune cells, and digested lipids and return these bodily fluids to blood through two pairs of lymphovenous valves (LVVs). Like other cardiovascular valves LVVs prevent the backflow of blood into the lymphatic vessels. In addition to LVVs, platelets are necessary to prevent the entry of blood into the lymphatic vessels. Platelet thrombi are observed at LVVs suggesting that LVVs and platelets function in synergy to regulate blood/lymphatic separation. OBJECTIVES: The primary objective of this work is to determine whether platelets can regulate blood/lymph separation independently of LVVs. METHODS: The transcription factor GATA2 is necessary for the development of both LVVs and hematopoietic stem cells. Using various endothelial- and hematopoietic cell expressed Cre-lines, we conditionally deleted Gata2. We hypothesized that this strategy would identify the tissue- and time-specific roles of GATA2 and reveal whether platelets and LVVs can independently regulate blood/lymph separation. RESULTS: Lymphatic vasculature-specific deletion of Gata2 results in the absence of LVVs without compromising blood/lymph separation. In contrast, deletion of GATA2 from both lymphatic vasculature and hematopoietic cells results in the absence of LVVs, reduced number of platelets and blood-filled lymphatic vasculature. CONCLUSION: GATA2 promotes blood/lymph separation through platelets. Furthermore, LVVs are the only known sites of interaction between blood and lymphatic vessels. The fact that blood is able to enter the lymphatic vessels of mice lacking LVVs and platelets indicates that under these circumstances the lymphatic and blood vessels are connected at yet to be identified sites.

Kinesin associated protein, DmKAP, binding harnesses the C-terminal ends of the Drosophila kinesin-2 stalk heterodimer.

The heterotrimeric kinesin-2 consists of two distinct motor subunits and an accessory protein, KAP, which binds to the coiled-coil stalk domains and one of the tail domains of the motor subunits. Genetic studies revealed that KAP is essential for the kinesin-2 functions in cilia, flagella, and axon. However, the structural significance of the KAP binding on kinesin-2 assembly and stability is not known. Here, using the Fluorescence Lifetime assay, we show that DmKAP binding selectively reduces the distance between the C-terminal ends of Drosophila kinesin-2 stalk heterodimer. Insertion of a missense mutation (E551K) in the Drosophila kinesin-2α stalk fragment, which was shown to reduce the structural dynamics of the stalk heterodimer earlier, also reduced the distances at both the N- and C-terminal ends of the stalk heterodimer independent of DmKAP. The zipping effect, particularly at the N-terminal end of the mutant stalk heterodimer, is further enhanced in the presence of DmKAP. Together, these results suggest that the KAP binding could alter the structural dynamics of kinesin-2 stalk heterodimer at the C-terminal end and stabilize the association between the stalk domains.

The structural dynamics of the kinesin-2 stalk heterodimer and its biological relevance.

Association between two motor subunits through the rod/stalk domain enables molecular motors to walk processively on protein filaments. Previous studies suggested that structural flexibility in the coiled-coil stalk of kinesins is essential for processive runs. The stalk of heterotrimeric kinesin-2, a comparatively less processive motor, is unstable at ambient temperature. How this structural instability impacts the motor function is unclear. Here, using the Förster Resonance Energy Transfer based assays, we show that the Drosophila kinesin-2α/ß stalk heterodimer is dynamic at physiological conditions. We further show that insertion of a missense mutation (Glu551-Lys) at the C-terminal half of kinesin-2α stalk reduces the dynamics of the heterodimeric stalk in vitro. The mutation, isolated as a recessive lethal allele in a forward genetic screen, is reported to disrupt the motor function in axonal transport and cilia development. Together these two results suggest that the dynamic instability of the kinesin-2 stalk could play a crucial role in maintaining its biological function.