Journal: bioRxiv

Article Title: Modification of the Neck Linker of KIF18A Alters Microtubule Subpopulation Preference

doi: 10.1101/2023.05.02.539080

Figure Lengend Snippet: (a) Cartoon of KIF18A dimer with important structural regions noted and sequence alignment of KIF18A homologs indicating conservation of S357 site. S.C.: Saccharomyces cerevisiae. (b) Representative immunofluorescence images of GFP KIF18A wild-type (WT), S357A, and S357D localization in bipolar HeLa Kyoto and RPE1 cells. Cells were fixed approximately 24 hours after knockdown of endogenous KIF18A and induction of GFP-KIF18A with doxycycline. White arrows indicate altered localization of KIF18A S357D. Brightness/contrast levels set differently to optimize visualization. Scale bar is 10 μm. (c) Representative immunofluorescence images of GFP-KIF18A localization in monopolar mitotic cells. Cells were fixed approximately 24 hours after siRNA treatment to knockdown endogenous KIF18A and induction of GFP-KIF18A with doxycycline. 2–3 hours prior to fixation, monopolar mitotic cells were established with addition of 100 μm monastrol. Brightness/contrast levels set differently to optimize visualization. WT: wild-type. Scale bar is 10 μm. (d) Representative immunofluorescence images of KIF18A wild-type (WT) and S357D localization in interphase cells. Cells were imaged live approximately 24 hours after siRNA treatment to knockdown endogenous KIF18A and induction of GFP-KIF18A with doxycycline. Colors indicate pseudo-color in merged image. Brightness/contrast levels set differently to optimize visualization. Cyan arrows indicate WT KIF18A localization at periphery of interphase cells, white arrows indicate additional accumulation of KIF18A S357D at periphery. Insets created with increased brightness and contrast to highlight peripheral localization. Scale bar is 100μm. WT: wild-type.

Article Snippet: Genomic DNA was extracted from resulting HeLa Kyoto and RPE1 inducible cell lines (QIAmp DNA Blood Mini Kit Qiagen #51106) and sequenced through the S357 mutation site (Eurofins) to verify correct incorporation of the desired construct.

Techniques: Sequencing, Immunofluorescence, Knockdown

Journal: bioRxiv

Article Title: Modification of the Neck Linker of KIF18A Alters Microtubule Subpopulation Preference

doi: 10.1101/2023.05.02.539080

Figure Lengend Snippet: (a) Representative DIC images highlighting quantification of nuclear envelope breakdown (NEB) to anaphase onset (AO). Blue arrows indicate DNA location in cell. (b-c) Quantification of NEB to AO (mitotic timing) in HeLa Kyoto cells (b) and RPE1 cells (c) . Horizontal line represents median. Each solid small dot represents a single cell, larger open circles represent the average from each experimental replicate. Data were acquired from three experimental replicates and were analyzed using a Kruskal-Wallis with Dunn’s Multiple Comparisons Test. P value style: P value style: <0.05 (*), if no significance is indicated result was not significant (> 0.05). Numbers above each condition indicate the median mitotic timing value. WT: wild-type, KD: knockdown. (d-e) Quantification of the percent of cells that never divide in HeLa Kyoto cells (d) and RPE1 cells (e) . Bar indicates mean, each dot represents the percentage of cells that never divided for an individual experimental replicate. Numbers above graphs indicate the mean overall value for each condition. WT: wild-type, KD: knockdown.

Article Snippet: Genomic DNA was extracted from resulting HeLa Kyoto and RPE1 inducible cell lines (QIAmp DNA Blood Mini Kit Qiagen #51106) and sequenced through the S357 mutation site (Eurofins) to verify correct incorporation of the desired construct.

Techniques: Knockdown

Journal: bioRxiv

Article Title: Modification of the Neck Linker of KIF18A Alters Microtubule Subpopulation Preference

doi: 10.1101/2023.05.02.539080

Figure Lengend Snippet: (a) Quantification of the ratio of peripheral EB1 signal to inner (spindle) EB1 signal. Solid horizontal line indicates mean, vertical lines indicate standard deviation. Each solid small dot represents a single cell, larger open dots represent the mean from each experimental replicate. Data were acquired from three experimental replicates and were analyzed using a one-way ANOVA with Tukey’s test for multiple comparisons. P value style: >0.05 (ns), if <0.05 (*). WT: wild-type. (b) Quantification of the ratio of peripheral GFP signal to inner (spindle) GFP signal. Solid horizontal line indicates mean, vertical lines indicate standard deviation. Each solid small dot represents a single cell, larger open dots represent the mean from each experimental replicate. Data were acquired from three experimental replicates and were analyzed using a one-way ANOVA with Tukey’s test for multiple comparisons. P value style: >0.05 (ns), if <0.05 (*). WT: wild-type. (c) Representative immunofluorescence images of EB1 and GFP in RPE1 cells. Cells were fixed approximately 24 hours after siRNA treatment to knockdown endogenous KIF18A and induction of GFP-KIF18A with doxycycline. 2–3 hours prior to fixation monopolar mitotic cells were established with addition of 100 μm monastrol. Color indicates pseudo-color in merged image. Brightness/contrast levels for GFP and EB1 are set to be equivalent, brightness/contrast levels for poles and DNA are set differently to optimize visualization. Scale bar is 10 μm.

Article Snippet: Genomic DNA was extracted from resulting HeLa Kyoto and RPE1 inducible cell lines (QIAmp DNA Blood Mini Kit Qiagen #51106) and sequenced through the S357 mutation site (Eurofins) to verify correct incorporation of the desired construct.

Techniques: Standard Deviation, Immunofluorescence, Knockdown

Journal: bioRxiv

Article Title: Modification of the Neck Linker of KIF18A Alters Microtubule Subpopulation Preference

doi: 10.1101/2023.05.02.539080

Figure Lengend Snippet: (a) Graphic demonstrating quantification of spindle and peripheral tubulin. (b) Representative images of tubulin, DNA, and GFP in RPE1 cells. Cells were fixed approximately 24 hours after siRNA treatment to knockdown endogenous KIF18A and induction of GFP-KIF18A with doxycycline. Color indicates pseudo-color in merged image. Brightness/contrast levels for tubulin are set to be equivalent, brightness/contrast levels for DNA and GFP are set differently to optimize visualization. Scale bar is 10 μm. WT: wild-type, KD: knockdown. (c-d) Quantification of background subtracted spindle tubulin (c) and peripheral tubulin (d) intensity in RPE1 cells. Horizontal line represents median. Each dot represents a single cell. Data were acquired from a minimum of three experimental replicates and were analyzed using a Kruskal-Wallis with Dunn’s Multiple Comparisons Test. P value style: <0.05 (*), if no significance is indicated result was not significant (> 0.05). WT: wild-type, KD: knockdown.

Article Snippet: Genomic DNA was extracted from resulting HeLa Kyoto and RPE1 inducible cell lines (QIAmp DNA Blood Mini Kit Qiagen #51106) and sequenced through the S357 mutation site (Eurofins) to verify correct incorporation of the desired construct.

Techniques: Knockdown

Journal: bioRxiv

Article Title: Modification of the Neck Linker of KIF18A Alters Microtubule Subpopulation Preference

doi: 10.1101/2023.05.02.539080

Figure Lengend Snippet: (a) KIF18A wild-type, shortened neck-linker (sNL), and S357D sequences. Blue text indicates neck-linker region. Red text indicates location of S357D mutation. (b) Quantification of GFP-KIF18A localization in periphery versus on kinetochore microtubules in mitotic cells. Solid horizontal line indicates mean, vertical lines indicate standard deviation. Each solid small dot represents a single cell. Black outlined dots indicate presence of HURP, gray outlined dots indicate HURP knockdown. Data were acquired from three experimental replicates and were analyzed using a one-way ANOVA with Tukey’s test for multiple comparisons. P value style: <0.05 (*), if no significance is indicated comparison was not significant. WT: wild-type. (c) Representative images of KIF18A localization in RPE1 cells. Cells were fixed approximately 24 hours after siRNA treatment to knockdown endogenous KIF18A and HURP and induction of GFP-KIF18A with doxycycline. Color indicates pseudo-color in merged image. +/− indicates presence or absence of HURP respectively. Brightness/contrast levels for HURP are set to be equivalent, brightness/contrast levels for kinetochores, DNA, and GFP are set differently to optimize visualization. Scale bar is 10 μm. WT: wild-type, sNL: shortened neck-linker, KD: knockdown.

Article Snippet: Genomic DNA was extracted from resulting HeLa Kyoto and RPE1 inducible cell lines (QIAmp DNA Blood Mini Kit Qiagen #51106) and sequenced through the S357 mutation site (Eurofins) to verify correct incorporation of the desired construct.

Techniques: Mutagenesis, Standard Deviation, Knockdown, Comparison