No Evidence for Localization of Bazooka/Par-3 to the Nucleus and Neuromuscular Junction in Drosophila

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Bazooka/Par-3 does not localize to the nuclear envelope or neuromuscular junction in Drosophila, contrary to previous reports.

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The study re-assessed the subcellular localization of the polarity regulator Bazooka/Par-3 (Baz) in Drosophila tissues. Initial immunostaining analysis using various anti-Baz antibodies revealed Baz localization at the nuclear envelope and neuromuscular junction (NMJ), consistent with previous reports. However, further analysis using Baz mutant clones and Baz-GFP fusion proteins showed that the nuclear envelope and NMJ staining patterns were artifacts not reflecting the true Baz localization.
The authors found that the nuclear envelope staining was observed in specific cell types, particularly polyploid cells undergoing endoreduplication. However, this staining persisted even in Baz mutant cells, indicating it was not specific to Baz. Similarly, the NMJ staining was unaffected by Baz RNAi, contradicting the previously reported NMJ phenotypes in Baz mutants.
The authors conclude that Baz does not actually localize to the nuclear envelope or NMJ, and the previously reported localization and functions of Baz at these sites should be interpreted with caution. The study highlights the importance of validating antibody specificity, especially when investigating subcellular localization, and using complementary approaches like tagged fusion proteins to confirm findings.

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Belangrijkste Inzichten Gedestilleerd Uit

Re-assessment of the subcellular localization of Bazooka/Par-3 in Drosophila: No evidence for localization to the nucleus and the neuromuscular junction

by Kim,S., Shah... om www.biorxiv.org 12-21-2023

https://www.biorxiv.org/content/10.1101/2023.12.21.572772v1

Diepere vragen

What other subcellular compartments or structures might Bazooka/Par-3 localize to and regulate in Drosophila cells?

Bazooka/Par-3 is known to play a crucial role in establishing and maintaining cell polarity in various cell types in Drosophila. Apart from the cell cortex and intercellular junctions where Baz is commonly localized, there are other subcellular compartments or structures where Bazooka/Par-3 might also localize and regulate cellular processes. One potential subcellular compartment where Baz could localize is the cytoplasmic membrane, particularly in regions involved in cell-cell adhesion and signaling. Baz has been shown to interact with other polarity proteins at the cell membrane to regulate cell polarity. Additionally, Baz may also localize to endomembrane compartments such as the endoplasmic reticulum or Golgi apparatus to regulate protein trafficking and secretion. Furthermore, Baz localization to organelles like the mitochondria or the centrosome could also be possible, as these organelles play essential roles in cell division and intracellular signaling pathways. Overall, Bazooka/Par-3 may have diverse subcellular localization patterns in Drosophila cells to regulate various cellular processes beyond cell polarity.

How can the previously reported NMJ phenotypes in Baz mutants be reconciled with the lack of Baz localization at the NMJ observed in this study?

The previously reported neuromuscular junction (NMJ) phenotypes in Baz mutants, such as reduced staining intensity for α-Spectrin and decreased number of synaptic boutons, may be attributed to secondary site mutations present in the baz alleles used in those studies. It is possible that these secondary mutations affect proteins or pathways related to NMJ development and function, leading to the observed phenotypes. Additionally, the reported NMJ phenotypes could be influenced by genetic background effects or interactions with other signaling pathways that indirectly impact NMJ structure and function. Therefore, the lack of Baz localization at the NMJ observed in this study suggests that Baz may not directly regulate NMJ development or maintenance. Instead, the NMJ phenotypes seen in Baz mutants in previous studies may be due to genetic interactions or secondary mutations that affect NMJ components independently of Baz localization.

Could the nuclear envelope staining observed with anti-Baz antibodies be due to cross-reactivity with an unidentified epitope, and if so, what is the identity of the true target protein?

The nuclear envelope staining observed with anti-Baz antibodies in this study is likely due to cross-reactivity with an unidentified epitope rather than specific localization of Bazooka/Par-3 to the nuclear envelope. The lack of Baz localization at the nuclear envelope in mutant clones and GFP-tagged Baz lines, along with the persistence of staining in mutant cells, suggests that the observed staining is not specific to Baz. The true target protein responsible for the nuclear envelope staining remains unidentified, but it could be a nuclear envelope component or a protein with a similar epitope to Baz that is recognized by the antibodies used in the immunostaining. Further investigation is needed to identify the exact protein or structure that is being detected by the anti-Baz antibodies at the nuclear envelope and to clarify the mechanism of cross-reactivity leading to this staining pattern.