Research Roundup: cell biology
Do you love cell biology? Here for you are three new research studies showing our antibodies in action. This latest Research Roundup talks about kinesin’s motor domain in microtubules, membranes & trafficking in organelles, and lysosomal protein composition.
Primary antibodies are effective tools to analyze living cell components at the molecular level and identify proteins that may be involved in or cause disease. For our Research Roundup series, we present you with three studies that have used our primary antibodies to better understand the role of kinesin’s motor domain in microtubules, membranes & trafficking in organelles, and lysosomal protein composition. Read on!
1. The motor domain of testis-enriched kinesin KIF9 is essential for its localization in the mouse flagellum (Miyata et al., 2021)
Kinesin is a molecular motor that moves along the microtubules carrying proteins up and down the microtubules. The kinesin family of microtubule motors is divided into subfamilies based on structure and function. For example, KIF9 is the founder of the Kinesin-9 subfamily.
In mice, KIF9 is testis-enriched, particularly in the mouse sperm flagellum. Deletion of the Kif9 gene results in male subfertility. However, it is still unclear if the motor domain of KIF9 is involved in normal sperm motility and male fertility.
To answer this question, this study used CRISPR/Cas9 system in mice to insert a mutation in a critical amino acid of the KIF9 motor domain, thus impairing kinesin’s motor activity. Specifically, the threonine of the ATP binding motif in the KIF9 motor domain was changed to asparagine (T100N).
The results show that T100N mutant mice exhibit reduced sperm motility and male fertility consistent with Kif9 knockout mice, clearly suggesting that the motor domain of KIF9 is essential for its localization in the sperm flagellum.
Since proteins of the axonemal cytoskeleton act as a scaffolding for various protein complexes and provide binding sites for molecular motor proteins such as kinesins, our rabbit polyclonal antibodies targeting axonemal proteins were used in western blot: anti‐DNAH17 (HPA024354), anti-GAS8 (HPA041311), and anti‐RSPH9 (HPA031703).
Figure 1. KIF9 is depleted in the spermatozoa of Kif9 T100N mutant mice. (A) Protein expression of KIF9 in testis and cauda epididymal spermatozoa. (B) ICC-IF of spermatids from Kif9 wild type and Kif9 T100N mutant mice. KIF9 is visible in green, acetylated tubulin in red, and nuclei in blue. (C) WB analysis of axonemal proteins in Kif9 wild type and Kif9 T100N mutant. Image from Miyata et al, 2021.
2. CLN6 deficiency causes selective changes in the lysosomal protein composition (Tuermer et al., 2021)
Neuronal ceroid lipofuscinosis (NCL), commonly known as Batten disease, refers to a group of conditions that affect the nervous system characterized by the accumulation of autofluorescent ceroid lipopigments in lysosomes. NCLs are accompanied by seizures, blindness, and premature mortality.
It is known that defects in several genes affecting various proteins lead to NCL, one of them being CLN6 (specifically involved in the childhood variant of NCL). However, the function of CLN6 and how its deficiency affects lysosomal integrity remains unknown.
This study applies a proteomic approach to run for the first time a quantitative comparison between the lysosomal proteome of Cln6-deficient mice and wild-type mice.
The analysis reveals that among the most reduced lysosomal proteins in Cln6-deficient liver lysosomes are the proteases that belong to the family of NCL proteins themselves, namely tripeptidylpetidase 1 (Tpp1, Cln1), palmitoyl-protein thioesterase 1 (Ppt1, Cln2), Cathepsin F (Ctsf, Cln13) and Cathepsin D (Ctsd, Cln10).
Our anti-Ppt1 polyclonal antibody (HPA021546) was used in WB to verify differences in lysosomal protein amounts.
Figure 2. Verification of differences in lysosomal protein amounts. Equal protein amounts of tritosome/lysosome fraction were separated by SDS-PAGE, blotted on nitrocellulose membrane, and probed with antibodies against different lysosomal proteins. Shown are three biological replicates each. hc = heavy chain, lc = light chain of fully mature Cathepsin D. Cropped image from Tuermer et al., 2021.
3. NPC1 regulates the distribution of phosphatidylinositol 4-kinases at Golgi and lysosomal membranes (Kutchukian et al., 2021)
Cholesterol and phosphoinositides (PI) are two critically essential lipids that are found in cellular membranes and dysregulated in many disorders. Therefore, uncovering molecular pathways connecting these essential lipids may offer new therapeutic insights.
This study reports that in Niemann-Pick Type C1 (NPC1) disease, a pathological neurodegenerative disorder of altered cholesterol homeostasis, there is a rearrangement of molecular components of the cholesterol/PtdIns4P cycle.
In particular, the authors identified a novel signaling axis that connects NPC1 cholesterol efflux to the regulation of Golgi and lysosomal PtdIns4P levels.
The results demonstrate that disease mutations or loss of lysosomal NPC1 function lead to the aberrant accumulation of PtdIns4P on Golgi and lysosomal membranes.
Our anti-OSBP (HPA039227) and anti-SAC1 (HPA069869) rabbit polyclonal antibodies were used in WB and immunofluorescence for detecting OSBP and SAC1 proteins, which serve as important regulators of PtdIns4P homeostasis.
Figure 3. NPC1 regulates the contents of ER-TGN membrane contact sites. (A) Diagram of the molecular elements within ER-TGN membrane contact sites. (B) Representative Western blot for OSBP in control and U18-treated tsA201 cells. Protein levels were determined by densitometry with b-actin normalization. Each point (n = 3) represents protein levels from U18-treated cells normalized to control bands—cropped image from Kutchukian et al., 2021.
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