CSLD5 Antibody

What is CSLD5 and what is its primary function in plant cells?

CSLD5 is a member of the Cellulose Synthase-Like D family that provides essential cell wall synthase activity during cytokinesis in plants. It functions specifically in the construction of newly forming cell plates during cell division . Unlike other family members that participate in both cell division and expansion processes, CSLD5 appears to be specialized for cell division activities. Research has shown that CSLD5 is uniquely enriched in rapidly dividing cell populations, particularly in the self-renewing meristemoid population in the stomatal lineage . It contributes to proper cell wall formation during cytokinesis, ensuring complete separation of daughter cells.

How is CSLD5 expressed and regulated during plant development?

CSLD5 expression is directly regulated by SPEECHLESS (SPCH), the master transcriptional regulator of stomatal lineage divisions. Chromatin immunoprecipitation experiments have confirmed that SPCH binds to proximal regions in the CSLD5 5′ regulatory region, and CSLD5 expression is upregulated upon SPCH induction . This regulation pattern explains why CSLD5 is enriched in dividing cells of the stomatal lineage. Furthermore, CSLD5 expression appears to be cell cycle-dependent, with protein accumulation occurring after the metaphase-anaphase transition during mitosis, as demonstrated by its temporally distinct expression pattern compared to cell cycle markers like CYCB1:1 .

What phenotypes are observed in csld5 mutant plants?

Plants with csld5 mutations display several distinct phenotypes:

These phenotypes are enhanced in double mutants with other CSLD family members (csld2/5 and csld3/5), indicating functional redundancy within the CSLD family . Interestingly, cell polarization is not significantly impaired in csld5 mutants, suggesting that the primary defect is related to cell wall formation rather than cell polarity establishment.

How does CSLD5 protein localization change during the cell cycle?

CSLD5 protein demonstrates remarkable cell cycle-dependent localization dynamics:

• CSLD5 is largely undetectable during S-phase and early mitosis

• Significant CSLD5 levels only appear after chromatin separation and initiation of cytokinesis

• CSLD5 specifically accumulates at the forming cell plate during cytokinesis

• The protein is rapidly lost once the new cell plate separates daughter cells

Time-lapse imaging reveals that Cerulean-CSLD5 fluorescence is observed only after separation of CYCB1:1-GFP labeled chromatin, with the proteins showing mutually exclusive expression patterns . Experiments with aphidicolin (a DNA polymerase inhibitor that arrests cells in S-phase) confirm this timing, as cells trapped in S-phase accumulate CYCB1:1-GFP but show undetectable levels of Cerulean-CSLD5 .

What is the molecular mechanism of CSLD5 protein turnover?

CSLD5 is an unstable protein that undergoes rapid degradation upon completion of cell division, unlike other CSLD family members and the closely related CESA family of cellulose synthases . Evidence suggests its degradation is regulated by a cell cycle-associated E3-ubiquitin ligase, likely the anaphase-promoting complex. This is supported by observations that CSLD5 protein turnover characteristics are altered in ccs52a2 mutants . The precise molecular triggers that mark CSLD5 for degradation during late cytokinesis remain an active area of research, but the tight temporal control ensures CSLD5 activity is restricted to cell division events.

How do CSLD5 and other CSLD family members functionally interact during cytokinesis?

The functional relationship between CSLD family members is complex:

• While CSLD2 and CSLD3 play roles in root hair development, CSLD5 does not appear to contribute to this process

• CSLD5 functions predominantly in cell division rather than expansion

• Double mutants (csld2/5 and csld3/5) show enhanced cytokinesis defects compared to single mutants, indicating partial functional redundancy

• All three proteins (CSLD2, CSLD3, and CSLD5) localize to forming cell plates during cytokinesis, suggesting overlapping functions in this process

Research indicates that CSLD5 has a specialized role in cytokinesis that cannot be fully compensated by other family members, explaining why csld5 mutants display specific cell division defects despite the presence of other CSLD proteins.

What are optimal approaches for visualizing CSLD5 during cell division?

For effective visualization of CSLD5 during cell division, researchers should consider:

• Using N-terminal fluorescent protein fusions (such as Cerulean-CSLD5) expressed under native promoters

• Co-visualization with cell cycle markers like CYCB1:1-GFP to precisely time CSLD5 appearance during mitosis

• Time-lapse imaging of dividing cells to capture the dynamic appearance and disappearance of CSLD5

• Complementation testing to confirm functionality of fluorescent fusions

One effective approach demonstrated in the literature involves creating a Cerulean-CSLD5 construct under the control of its endogenous promoter and confirming functionality through genetic rescue experiments. This allows for live imaging of protein dynamics while ensuring normal protein function is maintained .

How can researchers effectively use CSLD5 antibodies in experimental applications?

When working with CSLD5 antibodies for research applications:

• Select antibodies validated for specific applications (ELISA, Western blot) as indicated in product documentation

• Use appropriate positive controls, such as recombinant CSLD5 protein (some commercial antibodies include antigen as positive control)

• Include pre-immune serum as a negative control to assess non-specific binding

• Consider species specificity - antibodies are available for both Arabidopsis thaliana and Oryza sativa CSLD5

For Western blot applications, researchers should optimize protein extraction conditions considering CSLD5's membrane localization and rapid turnover characteristics. For immunolocalization studies, fixation procedures should preserve cell plate structures where CSLD5 accumulates during cytokinesis.

What experimental designs best elucidate CSLD5 function in stomatal development?

To investigate CSLD5's role in stomatal development, consider these experimental approaches:

• Quantitative analysis of stomatal clustering phenotypes in single (csld5) and double mutants (csld2/5, csld3/5)

• Time-lapse imaging of stomatal lineage divisions using fluorescently-tagged CSLD5 and polarity markers (e.g., BASL)

• Cell wall staining techniques to visualize incomplete cell walls in mutant backgrounds

• ChIP assays to confirm direct regulation by SPCH and identify other potential transcriptional regulators

Comparative analysis of cell division rates and patterns between wild-type and mutant plants, particularly in stomatal lineage cells, can provide valuable insights into CSLD5's specific contributions to this developmental process.

How can researchers address the low penetrance of csld5 cell wall division defects?

The csld5 mutation produces cell wall defects with relatively low penetrance (approximately 1-3%) , which can make phenotypic analysis challenging. To address this:

• Increase sample sizes substantially to ensure statistical significance

• Consider using sensitized genetic backgrounds (e.g., stomatal lineage) where CSLD5 defects are more pronounced

• Employ double mutants (csld2/5 or csld3/5) which enhance the penetrance of cytokinesis defects

• Use high-resolution time-lapse imaging to capture rare cell division events that result in defects

Despite low penetrance in single mutants, the defects observed in csld5 plants provide valuable information about the protein's function when properly quantified and analyzed.

What controls are essential when studying cell cycle-dependent CSLD5 expression?

When investigating CSLD5's cell cycle-dependent expression and localization:

• Include cell cycle stage markers (like CYCB1:1-GFP) for precise temporal correlation

• Use cell cycle inhibitors (e.g., aphidicolin) to synchronize cells and confirm stage-specific expression

• Employ membrane markers (like BRI1-GFP) to distinguish between cell plate formation defects and CSLD5 localization issues

• Compare CSLD5 dynamics with other CSLD family members as internal controls

These controls help distinguish between general cytokinesis mechanisms and CSLD5-specific functions, enabling more precise interpretation of experimental results.

What are promising approaches for identifying CSLD5 interaction partners during cytokinesis?

To identify CSLD5 interaction partners during cytokinesis, researchers could:

• Perform immunoprecipitation-mass spectrometry of tagged CSLD5 from synchronized dividing cells

• Use proximity labeling approaches (BioID or TurboID) fused to CSLD5 to identify proteins in close proximity during cell division

• Conduct genetic screens for enhancers or suppressors of csld5 mutant phenotypes

• Investigate potential interactions with known cell plate formation machinery components

Understanding CSLD5's protein interaction network would provide valuable insights into how this enzyme is integrated into the broader cytokinesis machinery and regulated during cell division.

How might CSLD5 function be evolutionarily conserved across plant species?

CSLD5 homologs exist across plant species, including in rice (Oryza sativa) , suggesting evolutionary conservation of function. Future research should explore:

• Comparative functional analysis of CSLD5 homologs in diverse plant species

• Investigation of whether cell cycle-dependent regulation is conserved

• Analysis of species-specific adaptations in CSLD5 function or regulation

• Examination of CSLD5 roles in species with different cell division patterns or stomatal development pathways

Cross-species complementation experiments could determine the degree of functional conservation and reveal any species-specific adaptations in CSLD5 activity or regulation.