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First published online 17 January 2006
doi: 10.1242/jcs.02750

Journal of Cell Science 119, 452-458 (2006)
Published by The Company of Biologists 2006
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MKLP1 requires specific domains for its dendritic targeting

Xiaohui Xu, Cheng He, Zhaohuan Zhang and Yizhang Chen*

Department of Neurobiology, Institute of Neuroscience, Second Military Medical University, Shanghai 200433, China


Figure 1
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  		Fig. 1. Schematic diagram of the MKLP1 coding sequence and its deletion constructs. The first diagram shows the approximate functional domains of MKLP1, represented on the scale of MKLP1 1-856. Shown below are the diagrams of the constructs used in this study. Numbers on the right denote the numbers of amino acids in the mutation constructs; construct D456-710 has 254 aa deleted (from 456-710) compared with MKLP1 1-856. The enhanced green fluorescent protein (eGFP) tag fused to the N-terminus is shown in green.

 

Figure 2
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  		Fig. 2. Western blots of cells transfected with eGFP-tagged MKLP1 constructs. GFP, lanes 1 and 10; MKLP(1-856), lane 2; MKLP1(1-840), lane 3; MKLP1(1-710), lane 4; MKLP1(1-456), lane 5; MKLP1(162-856), lane 6; MKLP1(461-856), lane 7; MKLP1(711-856), lane 8; MKLP1(811-856), lane 9; MKLP1(461-710), lane 11; D461-710, lane 12; MKLP1(461-840), lane 13; MKLP1(711-840), lane 14.

 

Figure 3
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  		Fig. 3. Distribution of eGFP-tagged MKLP1(1-856) or MKLP1(1-840) in HEK293A cells. (Upper panels) eGFP-MKLP1(1-856) concentrated in the mid-body at the end of cytokinesis and associated with microtubules when expressed in HEK293A cells. Arrowheads indicate mid-body. (Lower panels) eGFP-MKLP1(1-840) bound easier to microtubules and accumulated at the periphery of the cell where the plus ends of microtubules were located. Arrowheads indicate accumulated eGFP. DNA in the nucleus was stained with Hoechst 33258 dye (blue). Bar, 25 µm.

 

Figure 4
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  		Fig. 4. Comparision of distribution of eGFP and MKLP1-eGFP in hippocampal neurons. Immunofluorescence microscopy images of eGFP and MAP2, and overlays. eGFP alone can be distributed to the axon, but eGFP-tagged MKLP1 construct was restricted to soma-dendrites. Arrowheads indicate axons. Dendrites are indicated by staining for MAP2. Bar, 25 µm.

 

Figure 5
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  		Fig. 5. Localization of MKLP1-deletion constructs containing N-terminal domains that had been transfected into hippocampal neurons. Arrowheads indicate the location of axons. Immunofluorescence microscopy images of cells expressing MKLP1-deletion mutants (GFP, green); MAP2 immunoreactivity in the same neurons (MAP2, red). Deletion constructs MKLP1(1-456), -(1-710) and -(1-840) showed a different dendritic localization than full-length MKLP1. Bar, 25 µm.

 

Figure 6
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  		Fig. 6. Localization of MKLP1 in hippocampal neurons at 7 DIV that have been transfected with constructs that contain only the C-terminal but differ in length. Construct MKLP1(811-856) showed no special location in neurons. Constructs MKLP1(711-856) and (461-856) show expression that is restricted to the nucleus. Construct MKLP1(162-856) partly recovered the dendritic targeting of MKLP1. Dendrites are indicated by MAP2 staining. Arrowheads indicate axons. Bar, 25 µm.

 

Figure 7
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  		Fig. 7. MKLP1 constructs MKLP1(461-840) and (711-840) were expressed in 7 DIV cultured hippocampal neurons. Dendrites are indicated by MAP2 staining. Arrowheads indicate axons. Bar, 25 µm.

 

Figure 8
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  		Fig. 8. MKLP1 constructs MKLP1(461-710) or D456-710 were expressed in 7 DIV cultured hippocampal neurons. Dendrites are indicated by MAP2 staining. Bar, 25 µm.

 

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© The Company of Biologists Ltd 2006